Dissecting succulence: Crassulacean acid metabolism and hydraulic capacitance are independent adaptations in Clusia leaves.

Succulence is found across the world as an adaptation to water-limited niches. The fleshy organs of succulent plants develop via enlarged photosynthetic chlorenchyma and/or achlorophyllous water storage hydrenchyma cells. The precise mechanism by which anatomical traits contribute to drought tolerance is unclear, as the effect of succulence is multifaceted. Large cells are believed to provide space for nocturnal storage of malic acid fixed by crassulacean acid metabolism (CAM), whilst also buffering water potentials by elevating hydraulic capacitance (CFT ). The effect of CAM and elevated CFT on growth and water conservation have not been compared, despite the assumption that these adaptations often occur together. We assessed the relationship between succulent anatomical adaptations, CAM, and CFT , across the genus Clusia. We also simulated the effects of CAM and CFT on growth and water conservation during drought using the Photo3 model. Within Clusia leaves, CAM and CFT are independent traits: CAM requires large palisade chlorenchyma cells, whereas hydrenchyma tissue governs interspecific differences in CFT . In addition, our model suggests that CAM supersedes CFT as a means to maximise CO2 assimilation and minimise transpiration during drought. Our study challenges the assumption that CAM and CFT are mutually dependent traits within succulent leaves.

Leaf vein density correlates with crassulacean acid metabolism, but not hydraulic capacitance, in the genus Clusia.

BACKGROUND AND AIMS: Many succulent species are characterized by the presence of Crassulacean acid metabolism (CAM) and/or elevated bulk hydraulic capacitance (CFT). Both CAM and elevated CFT substantially reduce the rate at which water moves through transpiring leaves. However, little is known about how these physiological adaptations are coordinated with leaf vascular architecture. METHODS: The genus Clusia contains species spanning the entire C3-CAM continuum, and also is known to have >5-fold interspecific variation in CFT. We used this highly diverse genus to explore how interspecific variation in leaf vein density is coordinated with CAM and CFT. KEY RESULTS: We found that constitutive CAM phenotypes were associated with lower vein length per leaf area (VLA) and vein termini density (VTD), compared to C3 or facultative CAM species. However, when vein densities were standardized by leaf thickness, this value was higher in CAM than C3 species, which is probably an adaptation to overcome apoplastic hydraulic resistance in deep chlorenchyma tissue. In contrast, CFT did not correlate with any xylem anatomical trait measured, suggesting CAM has a greater impact on leaf transpiration rates than CFT. CONCLUSIONS: Our findings strongly suggest that CAM photosynthesis is coordinated with leaf vein densities. The link between CAM and vascular anatomy will be important to consider when attempting to bioengineer CAM into C3 crops.

The why and how of sunken stomata: does the behaviour of encrypted stomata and the leaf cuticle matter?

BACKGROUND: Stomatal pores in many species are separated from the atmosphere by different anatomical obstacles produced by leaf epidermal cells, especially by sunken stomatal crypts, stomatal antechambers and/or hairs (trichomes). The evolutionary driving forces leading to sunken or 'hidden' stomata whose antechambers are filled with hairs or waxy plugs are not fully understood. The available hypothetical explanations are based mainly on mathematical modelling of water and CO2 diffusion through superficial vs. sunken stomata, and studies of comparative autecology. A better understanding of this phenomenon may result from examining the interactions between the leaf cuticle and stomata and from functional comparisons of sunken vs. superficially positioned stomata, especially when transpiration is low, for example at night or during severe drought. SCOPE: I review recent ideas as to why stomata are hidden and test experimentally whether hidden stomata may behave differently from those not covered by epidermal structures and so are coupled more closely to the atmosphere. I also quantify the contribution of stomatal vs. cuticular transpiration at night using four species with sunken stomata and three species with superficial stomata. CONCLUSIONS: Partitioning of leaf conductance in darkness (gtw) into stomatal and cuticular contributions revealed that stomatal conductance dominated gtw across all seven investigated species with antechambers with different degrees of prominence. Hidden stomata contributed, on average, less to gtw (approx. 70 %) than superficial stomata (approx. 80 %) and reduced their contribution dramatically with increasing gtw. In contrast, species with superficial stomata kept their proportion in gtw invariant across a broad range of gtw. Mechanisms behind the specific behaviour of hidden stomata and the multipurpose origin of sunken stomata are discussed.

Bracken fern does not diminish arbuscular mycorrhizal fungus inoculum potential in tropical deforested areas.

Tropical montane forests are threatened by uncontrolled fire events because of agricultural expansion. Consequently, deforested areas frequently are dominated by the bracken fern, Pteridium spp., for long periods, and forest regeneration is limited. Despite considerable research on bracken-dominated ecosystems, little is known about the relationship between bracken mycorrhizal fungi and tree seedlings. Arbuscular mycorrhizal fungi (AMF) form symbiotic relationships with terrestrial plants, providing nutrients and protection against pathogens and promoting seedling growth and establishment. Therefore, AMF inoculum have high potential for forest restoration programs. Here, we compare the species diversity of AMF spores, root colonization, and seedling growth of Clusia trochiformis 1 year after the addition of different liquefied root inocula: forest conspecific, forest heterospecific, and from Pteridium rhizomes. Thirteen morphospecies of arbuscular mycorrhizal fungi were identified on the roots of C. trochiformis, and Glomus spp. were the most abundant in all treatments. No differences were observed in spore species richness and diversity among treatments, but spore density was the highest subsequent to the Pteridium inoculum. There was no significant difference in mycorrhizal root colonization and seedling growth of C. trochiformis among inoculated treatments. We found a positive relation between root colonization and total biomass. This study shows that the AMF communities in bracken areas and forests present similar characteristics and that the bracken fern does not limit AMF inoculum potential, favouring seedling growth of Clusia.

New Polyprenylated Phloroglucinol and Other Compounds Isolated from the Fruits of Clusia nemorosa (Clusiaceae).

Clusia nemorosa has been widely used in folk medicine to treat various ailments, including headaches and inflammation. Investigation of the fruits of Clusia nemorosa (Clusiaceae) led to the isolation and characterization of a new phloroglucinol derivative, named 6S,8S,28S-nemorosic acid (1), together with seven known compounds: friedelin (2), ß-sitosterol (3), stigmasterol (4), ß-sitosterol glycoside (5), kaempferol (6), quercetin (7) and dimethyl citrate (8). The structures were determined by extensive 1D- and 2D-NMR, CD and MS spectroscopic analyses.

Leaf anatomical traits which accommodate the facultative engagement of crassulacean acid metabolism in tropical trees of the genus Clusia.

Succulence and leaf thickness are important anatomical traits in CAM plants, resulting from the presence of large vacuoles to store organic acids accumulated overnight. A higher degree of succulence can result in a reduction in intercellular air space which constrains internal conductance to CO2. Thus, succulence presents a trade-off between the optimal anatomy for CAM and the internal structure ideal for direct C3 photosynthesis. This study examined how plasticity for the reversible engagement of CAM in the genus Clusia could be accommodated by leaf anatomical traits that could facilitate high nocturnal PEPC activity without compromising the direct day-time uptake of CO2 via Rubisco. Nine species of Clusia ranging from constitutive C3 through C3/CAM intermediates to constitutive CAM were compared in terms of leaf gas exchange, succulence, specific leaf area, and a range of leaf anatomical traits (% intercellular air space (IAS), length of mesophyll surface exposed to IAS per unit area, cell size, stomatal density/size). Relative abundances of PEPC and Rubisco proteins in different leaf tissues of a C3 and a CAM-performing species of Clusia were determined using immunogold labelling. The results indicate that the relatively well-aerated spongy mesophyll of Clusia helps to optimize direct C3-mediated CO2 fixation, whilst enlarged palisade cells accommodate the potential for C4 carboxylation and nocturnal storage of organic acids. The findings provide insight on the optimal leaf anatomy that could accommodate the bioengineering of inducible CAM into C3 crops as a means of improving water use efficiency without incurring detrimental consequences for direct C3-mediated photosynthesis.

Facultative crassulacean acid metabolism (CAM) plants: powerful tools for unravelling the functional elements of CAM photosynthesis.

Facultative crassulacean acid metabolism (CAM) describes the optional use of CAM photosynthesis, typically under conditions of drought stress, in plants that otherwise employ C3 or C4 photosynthesis. In its cleanest form, the upregulation of CAM is fully reversible upon removal of stress. Reversibility distinguishes facultative CAM from ontogenetically programmed unidirectional C3-to-CAM shifts inherent in constitutive CAM plants. Using mainly measurements of 24h CO2 exchange, defining features of facultative CAM are highlighted in five terrestrial species, Clusia pratensis, Calandrinia polyandra, Mesembryanthemum crystallinum, Portulaca oleracea and Talinum triangulare. For these, we provide detailed chronologies of the shifts between photosynthetic modes and comment on their usefulness as experimental systems. Photosynthetic flexibility is also reviewed in an aquatic CAM plant, Isoetes howellii. Through comparisons of C3 and CAM states in facultative CAM species, many fundamental biochemical principles of the CAM pathway have been uncovered. Facultative CAM species will be of even greater relevance now that new sequencing technologies facilitate the mapping of genomes and tracking of the expression patterns of multiple genes. These technologies and facultative CAM systems, when joined, are expected to contribute in a major way towards our goal of understanding the essence of CAM.

The genome sequence of a druid fly, Clusia tigrina (Fallén, 1820).

We present a genome assembly from an individual male Clusia tigrina (a druid fly; Arthropoda; Insecta; Diptera; Clusiidae). The genome sequence is 1,216.4 megabases in span. Most of the assembly is scaffolded into 5 chromosomal pseudomolecules, including the X and Y sex chromosomes. The mitochondrial genome has also been assembled and is 17.68 kilobases in length.

Chemical characterization and antioxidant potential of ecuadorian propolis.

The chemical composition and the antioxidant potential of Ecuadorian propolis samples (n = 19) collected in different provinces were investigated. HPLC-DAD-ESI/MSn and GC-EI-MS analysis of the methanol extracts enabled us to define six types of Ecuadorian propolis based on their secondary metabolite composition. 68 compounds were identified, 59 of which are reported for the first time in Ecuadorian propolis. The detected compounds include flavonoids, diterpenes, triterpenes, organic acid derivatives, alkylresorcinol derivatives and nemorosone. Plants belonging to genera Populus, Mangifera and Clusia seemed to be vegetable sources employed by bees to produce Ecuadorian propolis. Total phenolic content and antioxidant activity of propolis extracts were determined by the Folin-Ciocalteu assay and 2,2-diphenyl-1-picrylhydrazyl and ferric reducing/antioxidant potential assays, respectively. As expected, the variable chemical composition affected the differences in terms of antioxidant potential.

Utilization of the Plant Clusia Fluminensis Planch & Triana Against Some Toxic Activities of the Venom of Bothrops jararaca and B. jararacussu Snake Venom Toxic Activities.

BACKGROUND: In Brazil, the Bothrops genus accounts for 87% of registered snakebites, which are characterized by hemorrhage, tissue necrosis, hemostatic disturbances, and death. The treatment recommended by governments is the administration of specific antivenoms. Although antivenom efficiently prevents venom-induced lethality, it has limited efficacy in terms of preventing local tissue damage. Thus, researchers are seeking alternative therapies able to inhibit the main toxic effects of venoms, without compromising safety. OBJECTIVE: The study aimed to test the ability of aqueous extracts of leaves, stems, and fruits of the plant Clusia fluminensis to neutralize some toxic effects induced by the venoms of Bothrops jararaca and Bothrops jararacussu. METHODS: The plant extracts were incubated with venoms for 30 min. at 25 °C, and then in vitro (coagulant and proteolytic) and in vivo (hemorrhagic, myotoxic, and edematogenic) activities were evaluated. In addition, the extracts were administered to animals (by oral, intravenous or subcutaneous routes) before or after the injection of venom samples, and then hemorrhage and edema assays were performed. In addition, a gel solution of the fruit extract was produced and tested in terms of reducing hemorrhage effects. A chemical prospection was performed to identify the main classes of compounds present in the extracts. RESULTS: All the extracts inhibited the activities of the two venoms, regardless of the experimental protocol or route of administration of the extracts. Moreover, the gel of the fruit extract inhibited the venom-induced-hemorrhage. The extracts comprised of tannins, flavonoids, saponins, steroids, and terpenoids. CONCLUSION: Antivenom properties of C. fluminensis extracts deserve further investigation in order to gain detailed knowledge regarding the neutralization profile of these extracts.

Effects of nemorosone, isolated from the plant Clusia rosea, on the cell cycle and gene expression in MCF-7 BUS breast cancer cell lines.

BACKGROUND: Breast cancer is the cause of considerable morbidity and mortality in women. While estrogen receptor antagonists have been widely used in breast cancer treatment, patients have increasingly shown resistance to these agents and the identification of novel targeted therapies is therefore required. Nemorosone is the major constituent of the floral resin from Clusia rosea and belongs to the class of polycyclic polyisoprenylated benzophenones of the acylphloroglucinol group. The cytotoxicity of nemorosone in human cancer cell lines has been reported in recent years and has been related to estrogen receptors in breast cancer cells. METHODS: Changes induced by nemorosone in the cell cycle and gene expression of the MCF-7 BUS (estrogen-dependent) breast cancer cell line were analyzed using flow cytometry and the RT(2) Profiler PCR array, respectively. RESULTS: In comparison to breast cancer cells without treatment, nemorosone induced discrete cell cycle arrest in the G1 phase and significant depletion in the G2 phase. Moreover, the compound altered the expression of 19 genes related to different pathways, especially the cell cycle, apoptosis and hormone receptors. CONCLUSION: These promising results justify further studies to clarify mechanisms of action of nemorosone, in view of evaluate the possible use of this benzophenone as adjuvant in the treatment of breast cancer.

Hydraulic architecture, water relations and vulnerability to cavitation of Clusia uvitana Pittier: a C3 -CAM tropical hemiepiphyte.

Clusia uvitana Pittíer (Clusiacea) is a tropical hemiepiphyte that has been shown to display a high plasticity in the expression of CAM in response to the environment. When water is available CO2 is taken up mostly during the- day. This study of the water relations and hydraulic architecture has revealed that leaf water potentials, £ ranged from 0-7 to -0.9 MPa and changed very little with time or water availability. The absolute hydraulic conductivity of stem segments (K,) and the specific conductivity (K1 ) were comparable to many other temperate and tropical species, but the leaf specificity conductivity (K1 ) was 1/3 to 1/30 that of many other species. So stems supported high leaf areas per unit of hydraulic conductivity. C uvitana was very vulnerable to cavitation, reaching 50 % loss of hydraulic conductivity at stem £=1.3 MPa. The species survives in spite of low K1 and high xylem vulnerability, because the CAM physiology insures low transpiration rates and high ability to evade dehydration.

Ecophysiological comportment of the tropical CAM-tree Clusia in the field: I. Growth of Clusia rosea Jacq. on St John, US Virgin Islands, Lesser Antilles.

Clusia rosea Jacq. is abundant in the moist parts of the Caribbean island of St John (US Virgin Islands, Lesser Antilles) but relatively rare along the dry south coast. Three types of seedlings were encountered, terrestrial seedlings, seedlings growing as humus-epiphytes on other trees, and seedlings growing inside the tanks of the bromeliad Aechmea lingulata (L.) Baker. Free-living trees grow from terrestrial seedlings or from epiphytic seedlings strangling and shading their host trees. Leaf-Na+ levels were always low (1-4 mequiv I-1 tissue water); trees close to the shore were not affected by salinity. In leaves of mature C. rosea trees, levels of Ca2+ , Mg2+ and K+ were about 60-90, 40-50, 45-55 mequiv I-1 tissue water, respectively. Epiphytic seedlings tended to contain lower levels of these inorganic cations than seedlings growing terrestrially or in the tanks of Ae. lingulata. Epiphytic seedlings contained significantly less nitrogen than terrestrial seedlings. In the leaves of mature trees N-levels were independent of altitude and location on the island, but shaded leaves had significantly higher N-levels than exposed leaves. Light compensation point of photosynthesis in epiphytic seedlings performing C3 -photosynthesis was 17-5 (µmol photons m-2 s-1 ), photosynthesis was saturated at about 300µmol photons m-2 s-1 showing a maximum rate of CO2 -uptake of 2-3 µmol m-2 s-1 .

Ecophysiological comportment of the tropical CAM-tree Clusia in the field: II. Modes of photosynthesis in trees and seedlings.

Measurements were performed on leaves of Clusia rosea Jacq. trees in the moist central mountains (330 to 365 m above sea level) and at the dry south coast of St John Island (US Virgin Islands, Lesser Antilles). Seedlings of C. rosea were also studied in the central hills. During the study period (March 1989) all trees showed crassulacean acid metabolism (CAM), in which net CO2 uptake extended for a remarkably long time in the morning (phase II of CAM: until about 11 to 12 h) and contributed about 1/3 of total net CO2 -uptake. During the night (phase I of CAM) malic acid and citric acid were accumulated concurrently at a molar ratio of malic: citric acid of about 1.6. Internal recycling of respiratory CO2 was 20% of total CO2 fixed during the night. Water-use-efficiency (mol CO2 taken up: mol H2 O transpired) was 0.014 to 0.022. The pH of leaf-cell sap at the end of the dark period was 2.85. This would still allow an H+ -ATPase at the tonoplast to transport 2H+ into the vacuole per ATP hydrolysed when operating near thermodynamic equilibrium. Free sugars, glucose and fructose, and starch were used as precursors for the CO2 -acceptor phosphoenolpyruvate during the dark period; contributions of the two hexoses were about equal and together four-times that of starch. Xylem tensions showed increases of up to 8 bar during day-time. Leaf-sap osmotic pressures did not change significantly; the trend was a small decline during day-time. Among the seedlings, three different modes of photosynthesis were encountered, namely C3 -photosynthesis in terrestrial and in epiphytic seedlings, continuous stomatal opening and CO2 -uptake day and night in epiphytic seedlings, and CAM in seedlings growing in the tanks of Aechmea lingulata (L.) Baker.

A one-year study on carbon, water and nutrient relationships in a tropical C3 -CAM hemi-epiphyte, Clusia uvitana Pittier.

Diel (24 h) courses of CO2 and water-vapour exchange of leaves of hemi-epiphytic plants of Clusia uvitana Pittier (Clusiaceae) were measured under natural tropical conditions in the semi-evergreen moist forest of Barro Colorado Island, Panama, from January 1991 until January 1992. Plants were studied at two sites, in the crown of a 47-m tall tree (Ceiba pentandra) and on the shore of Lake Gatun, at a height of about 2-4 m. The following results were obtained: (1) Diel carbon gain was mainly a function of photosynthetic photon fluence rate (PPFR) on individual days. PPFR also strongly affected CAM activity. A leafless period of the host tree Ceiba pentandra resulted in higher incident PPFRs and slightly lower nighttime temperatures in the canopy of C uvitana; this led to increases in both daytime and nighttime CO2 fixation. (2) In fully mature sun leaves from the two sites, nocturnal net uptake of atmospheric CO2 occurred on almost all of the 71 days measured and nocturnal carbon gain was enhanced during the dry season. (3) In C. uvitana at the Lake site, 24-h carbon gain during the wet and dry season was similar to C. uvitana at the Ceiba site during the leafless period of the host tree. Overall CAM activity was lower at the Lake site. (4) Recycling of respiratory CO2 was a major route for nocturnal acid synthesis. Nocturnal net uptake of atmospheric CO2 was closely correlated with changes in titratable acidity, but accounted for only about 30% of the nocturnal increase in organic acids. (5) Mature shade leaves performed CAM only during the dry season, whereas in the wet season they showed atmospheric CO2 uptake exclusively in the light. (6) Independent of exposure or season, leaves less than about 12 wk old showed a C3 pattern of diel gas exchange and the level of titratable acidity was high day and night. (7) The annual carbon budget of outer canopy leaves of C uvitana in the Ceiba site was 1780 g CO2 m-2 a-1 and the average long-term water-use efficiency was 23 × 10-3 g CO2 g-1 H2 O. (8) Vegetative growth was strongly seasonal. Branch length increment and leaf area development was much higher in the wet season. Mineral element contents in these evergreen plants showed no age-related changes, but a significant proportion of elements was retrieved before abscission.

Phosotynthesis in hemiepiphytic species of Clusia and Ficus.

Hemiepiphytic species in the genera Clusia and Ficus were investigated to study their mode of photosynthetic metabolism when growing under natural conditions. Despite growing sympatrically in many areas and having the same growth habit, some Clusia species show Crassulacean acid metabolism (CAM) whereas all species of Ficus investigated are C3. This conclusion is based on diurnal CO2 fixation patterns, diurnal stomatal conductances, diurnal titratable acidity fluctuations, and δ13C isotope ratios. Clusia minor, growing in the savannas adjacent to Barinas, Venezuela, shows all aspects of Crassulacean acid metabolism (CAM) on the basis of nocturnal gas exchange, stomatal conductance, total titratable acidity, and carbon isotope composition when measured during the dry season (February 1986). During the wet season (June 1986), the plants shifted to C3-type gas exchange with all CO2 uptake occurring during the daylight hours. The carbon isotope composition of new growth was-28 to-29‰ typical of C3 plants.

Light and dark CO2 fixation in Clusia uvitana and the effects of plant water status and CO2 availability.

In well-watered plants of Clusia uvitana, a species capable of carbon fixation by crassulacean acid metabolism (CAM), recently expanded leaves gained 5 to 13-fold more carbon during 12 h light than during 12 h dark periods. When water was withheld from the plants, daytime net CO2 uptake strongly decreased over a period of several days, whereas there was a marked increase in nocturnal carbon gain. Photosynthetic rates in the chloroplasts were hardly affected by the water stress treatment, as demonstrated by measurements of chlorophyll a fluorescence of intact leaves, indicating efficient decarboxylation of organic acids and refixation of carbon in the light. Within a few days after rewatering, plants reverted to the original gas exchange pattern with net CO2 uptake predominantly occurring during daytime. The reversible increase in dark CO2 fixation was paralleled by a reversible increase in the content of phosphoenolpyruvate (PEP) carboxylase protein. In wellwatered plants, short-term changes in the degree of dark CO2 fixation were induced by alterations in CO2 partial pressure during light periods: a decrease from 350 to 170 µbar CO2 caused nocturnal carbon gain, measured in normal air (350 µbar), to increase, whereas an increase to 700 µbar CO2, during the day, caused net dark CO2 fixation to cease. The increased CAM activity in response to water shortage may, at least to some extent, be directly related to the reduced carbon gain during daytime.

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

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 ([Formula: see text]) 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 [Formula: see text] 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.

Short-term changes in carbon-isotope discrimination in the C3-CAM intermediate Clusia minor L. growing in Trinidad.

On-line instantaneous carbon isotope discrimination was measured in conjunction with net uptake of CO2 in leaves of exposed and shaded plants of the C3-CAM intermediate Clusia minor growing under natural conditions in Trinidad. At the end of the rainy season (late January-early February, 1992) C3 photosynthesis predominated although exposed leaves recaptured a small proportion of respiratory CO2 at night for the synthesis of malic acid. Citric acid was the major organic acid accumulated by exposed leaves at this time with a citric: malic acid ratio of 11:1. Values of instantaneous discrimination (Δ) in exposed leaves during the wet season rose from 17.1‰ shortly after dawn to 22.7‰ around mid-day just before stomata closed, suggesting that most CO2 was fixed by Rubisco at this time. During the late afternoon, instantaneous Δ declined from 22.2‰ to 17‰, probably reflecting the limited contribution from PEPc activity and an increase in diffusional resistance to CO2 in exposed leaves. Shaded leaves showed no CAM activity and CO2 uptake proceeded throughout the day in the wet season. The decrease in instantaneous Δ from 27‰ in the morning to 19.2‰ in the late afternoon was therefore entirely due to diffusional limitation. Leaves sampled in the dry season (mid-March, 1992) had by now induced full CAM activity with both malic and citric acids accumulated overnight and stomata closed for 4-5 h over the middle of the day. Values of instantaneous Δ measured over the first 3 h after dawn (6.4-9.1‰) indicated that C4 carboxylation dominated CO2 uptake for most of the morning when rates of photosynthesis were maximal, implying that under natural conditions, the down regulation of PEPc in phase II occurs much more slowly than laboratory-based studies have suggested. The contribution from C3 carboxylation to CO2 uptake during phase II was most marked in leaves which accumulated lower quantities of organic acids overnight. In exposed leaves, measurements of instantaneous Δ during the late afternoon illustrated the transition from C3 to C4 carboxylation with stomata remaining open during the transition from dusk into the dark period. Uptake of CO2 by shaded leaves during the late afternoon however appeared to be predominantly limited by decreased stomatal conductance. The short-term measurements of instantaneous Δ were subsequently integrated over 24 h in order to predict the leaf carbon isotope ratios (δp) and to compare this with the δp measured for leaf organic material. Whilst there was close agreement between predicted and measured δp for plants sampled in the wet season, during the dry season the predicted carbon isotope ratios were 5-9‰ higher than the measured isotope ratios. During the annual cycle of leaf growth most carbon was fixed via the C3 pathway although CAM clearly plays an important role in maintaining photochemical integrity in the dry season.

Is crassulacean acid metabolism activity in sympatric species of hemi-epiphytic stranglers such as Clusia related to carbon cycling as a photoprotective process?

A comparison of carbon isotope discrimination characteristics, crassulacean acid metabolism (CAM) activity and gas exchange together with concurrent analysis of photosystem II (PSII) chlorophyll fluorescence was conducted on leaves of sympatric species of Clusia from the restinga of Barra de Maricá, Brazil. The carbon isotope discrimination (Δ) and leaf-sap titratable acidity for leaves collected in the field indicated that the carbon metabolism of one species, C. lanceolata, was predominantly C3-like, and a second, C. fluminensis, constitutive CAM. When well-watered under glasshouse conditions C. lanceolata displayed a gas exchange pattern expected of a C3 plant, where values of instantaneous discrimination (Δ) rose from 13.5% shortly after dawn to 21.9‰ at midday, suggesting that all CO2 uptake was mediated solely by ribulose 1,5-bisphosphate carboxylase (RUBISCO). C. fluminensis showed a gas exchange pattern which clearly exhibited all four phases of CAM. Δ values during phase II ranged from -0.4‰ at dawn to 5.9‰ some 3 h later, indicating that C4 carboxylation dominated CO2 uptake during the morning with an increasing contribution by RUBISCO, suggested by the 5‰ shift in Δ at this time. The dominance of phosphoenol-pyruvate carboxylase (PEPc) activity was also found during phase IV, and extended throughout the dark period (phase I) in C. fluminensis, such that values of Δ measured were negative (-5.0 to -0.4‰). This is the first time that negative Δ values have been reported, close to those predicted theoretically for PEPc activity. The day-time uptake of CO2 mediated by PEPc could lead to futile cycling through RUBISCO. In C. fluminensis organic acids were subjected to carbon turnover between PEPc and RUBISCO during phase II of CAM, serving perhaps to dissipate ATP and reductant at a time when excess photons are absorbed. Under low levels of photosynthetically active radiation (PAR) the two species displayed similar chlorophyll fluorescence characteristics, although for the CAM C. fluminensis a lower rate of decarboxylation of acids in the afternoon was reflected in changed quenching capacity. Under high PAR both species responded directly to changes in incident radiation, reflected by decreases in photon use efficiency (ΦPSII) and the intrinsic photochemical efficiency (F V/F M), together with high and reversible quenching of excess light by the means of radiationless or thermal dissipation (q N). Both species, with such markedly different carboxylation characteristics achieve similar rates of electron transport and maintain photosynthetic integrity. Under field conditions, however the severity of a prolonged dry season caused the CAM species to become deciduous, whereas the "C3-like" species remained healthy. This suggests that the widely expected advantages of CAM do not extend to tolerance of extreme environmental conditions, in contrast to the more C3-like of these sympatric species.