Dark CO2 Uptake Research Articles

Phosphorolytic degradation of leaf starch via plastidic α-glucan phosphorylase leads to optimized plant growth and water use efficiency over the diel phases of Crassulacean acid metabolism.

In plants with Crassulacean acid metabolism (CAM), it has been proposed that the requirement for nocturnal provision of phosphoenolpyruvate as a substrate for CO2 uptake has resulted in a re-routing of chloroplastic starch degradation from the amylolytic route to the phosphorolytic route. To test this hypothesis, we generated and characterized four independent RNAi lines of the obligate CAM species Kalanchoë fedtschenkoi with a >10-fold reduction in transcript abundance of plastidic α-glucan phosphorylase (PHS1). The rPHS1 lines showed diminished nocturnal starch degradation, reduced dark CO2 uptake, a reduction in diel water use efficiency (WUE), and an overall reduction in growth. A re-routing of starch degradation via the hydrolytic/amylolytic pathway was indicated by hyperaccumulation of maltose in all rPHS1 lines. Further examination indicated that whilst operation of the core circadian clock was not compromised, plasticity in modulating net dark CO2 uptake in response to changing photoperiods was curtailed. The data show that phosphorolytic starch degradation is critical for efficient operation of the CAM cycle and for optimizing WUE. This finding has clear relevance for ongoing efforts to engineer CAM into non-CAM species as a means of boosting crop WUE for a warmer, drier future.

Facultative crassulacean acid metabolism (CAM) in four small C3 and C4 leaf-succulents

Measurements of whole-plant gas exchange and titratable acidity demonstrate that the Australian native species Anacampseros australiana J.M.Black (Anacampserotaceae), Crassula sieberiana (Schult. & Schult.f.) Druce (Crassulaceae) and Portulaca australis Endl. (Portulacaceae) and the widespread naturalised tropical exotic, Portulaca pilosa L., exhibit facultative crassulacean acid metabolism (CAM). In well-watered plants, net CO2 uptake was restricted to the daylight hours and occurred via the C3 pathway (A. australiana and C. sieberiana) or the C4 pathway (P. australis and P. pilosa). Leaves of well-watered plants did not accumulate titratable acidity during the night. Following drought treatment, CO2 uptake in the light by shoots decreased markedly, nocturnal gas-exchange shifted from net CO2 loss to a CAM-type pattern that included net CO2 uptake, and leaves acidified at night. Nocturnal CO2 uptake by shoots and leaf acidification were most pronounced in A. australiana and least so in C. sieberiana. The induction of dark CO2 uptake and tissue acidification was fully reversible in all four species: upon rewatering, nocturnal CO2 uptake and acidification ceased and the rates of CO2 incorporation in the light were restored. We suggest that, hitherto considered relatively exceptional globally, facultative CAM may be more common than previously suspected, particularly among the generally small ephemeral leaf-succulents that characterise Australia’s succulent flora.

Aquatic CAM photosynthesis: A brief history of its discovery

Aquatic CAM (Crassulacean Acid Metabolism) photosynthesis was discovered while investigating an unrelated biochemical pathway concerned with anaerobic metabolism. George Bowes was a significant contributor to this project early in its infancy. Not only did he provide me with some valuable perspectives on peer review rejections, but by working with his gas exchange system I was able to take our initial observations of diel fluctuations in malic acid to the next level, showing this aquatic plant exhibited dark CO2 uptake. CAM is universal in all aquatic species of the worldwide Lycophyta genus Isoetes and non-existent in terrestrial Isoetes. Outside of this genus aquatic CAM has a limited occurrence in three other families, including the Crassulaceae. This discovery led to fascinating adventures in the highlands of the Peruvian Andes in search of Stylites, a terrestrial relative of Isoetes. Stylites is a plant that is hermetically sealed from the atmosphere and obtains all of its carbon from terrestrial sources and recycles carbon through CAM. Considering the Mesozoic origin of Isoetes in shallow pools, coupled with the fact that aquatic Isoetes universally possess CAM, suggests the earliest evolution of CAM photosynthesis was most likely not in terrestrial plants.

Soil Greenhouse Gas Fluxes during Wetland Forest Retreat along the Lower Savannah River, Georgia (USA)

Tidal freshwater forested wetlands (tidal swamps) are periodically affected by salinity intrusion at seaward transitions with marsh, which, along with altered hydrology, may affect the balance of gaseous carbon (C) and nitrogen (N) losses from soils. We measured greenhouse gas emissions (CO2, CH4, N2O) from healthy, moderately degraded, and degraded tidal swamp soils undergoing sea-level-rise-induced retreat along the lower Savannah River, Georgia, USA. Soil CO2 flux ranged from 90.2 to 179.1 mg CO2 m−2 h−1 among study sites, and was the dominant greenhouse gas emitted. CO2 flux differed among sites in some months, while CH4 and N2O fluxes were 0.18 mg CH4 m−2 h−1 and 1.23 μg N2O m−2 h−1, respectively, with no differences among sites. Hydrology, soil temperature, and air temperature, but not salinity, controlled the annual balance of soil CO2 emissions from tidal swamp soils. No clear drivers were found for CH4 or N2O emissions. On occasion, large ebbing or very low tides were even found to draw CO2 fluxes into the soil (dark CO2 uptake), along with CH4 and N2O. Overall, we hypothesized a much greater role for salinity and site condition in controlling the suite of greenhouse gases emitted from tidal swamps than we discovered, and found that CO2 emissions—not CH4 or N2O–contributed most to the global warming potential from these tidal swamp soils.

Isolation and characterization of mutants of common ice plant deficient in crassulacean acid metabolism.

Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that improves water use efficiency by shifting part or all of net atmospheric CO2 uptake to the night. Genetic dissection of regulatory and metabolic attributes of CAM has been limited by the difficulty of identifying a reliable phenotype for mutant screening. We developed a novel and simple colorimetric assay to measure leaf pH to screen fast neutron-mutagenized populations of common ice plant (Mesembryanthemum crystallinum), a facultative CAM species, to detect CAM-deficient mutants with limited nocturnal acidification. The isolated CAM-deficient mutants showed negligible net dark CO2 uptake compared with wild-type plants following the imposition of salinity stress. The mutants and wild-type plants accumulated nearly comparable levels of sodium in leaves, but the mutants grew more slowly than the wild-type plants. The mutants also had substantially reduced seed set and seed weight relative to wild type under salinity stress. Carbon-isotope ratios of seed collected from 4-month-old plants indicated that C3 photosynthesis made a greater contribution to seed production in mutants compared to wild type. The CAM-deficient mutants were deficient in leaf starch and lacked plastidic phosphoglucomutase, an enzyme critical for gluconeogenesis and starch formation, resulting in substrate limitation of nocturnal C4 acid formation. The restoration of nocturnal acidification by feeding detached leaves of salt-stressed mutants with glucose or sucrose supported this defect and served to illustrate the flexibility of CAM. The CAM-deficient mutants described here constitute important models for exploring regulatory features and metabolic consequences of CAM.

Environment or Development? Lifetime Net CO2 Exchange and Control of the Expression of Crassulacean Acid Metabolism inMesembryanthemum crystallinum

The relative influence of plant age and environmental stress signals in triggering a shift from C(3) photosynthesis to Crassulacean acid metabolism (CAM) in the annual halophytic C(3)-CAM species Mesembryanthemum crystallinum was explored by continuously monitoring net CO(2) exchange of whole shoots from the seedling stage until seed set. Plants exposed to high salinity (400 mm NaCl) in hydroponic culture solution or grown in saline-droughted soil acquired between 11% and 24% of their carbon via net dark CO(2) uptake involving CAM. In contrast, plants grown under nonsaline, well-watered conditions were capable of completing their life cycle by operating in the C(3) mode without ever exhibiting net CO(2) uptake at night. These observations are not consistent with the widely expressed view that the induction of CAM by high salinity in M. crystallinum represents an acceleration of preprogrammed developmental processes. Rather, our study demonstrates that the induction of the CAM pathway for carbon acquisition in M. crystallinum is under environmental control.

Expression of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxylase kinase genes. Implications for genotypic capacity and phenotypic plasticity in the expression of crassulacean acid metabolism.

In plants with crassulacean acid metabolism (CAM), dark CO2 uptake is mediated by phosphoenolpyruvate carboxylase (PEPC), an enzyme that can be regulated at transcriptional and posttranslational levels. Reversible phosphorylation of PEPC is catalyzed by a dedicated PEPC kinase, which in turn is regulated at the transcriptional level over the 24-h cycle in CAM plants. PEPC kinase controls the day/night regulation of PEPC during the CAM cycle, thus facilitating plasticity for optimizing CO2 uptake under different environmental conditions. To understand the importance of PEPC kinase in relation to its target PEPC in terms of CAM performance, the expression of the genes encoding the two enzymes was investigated in four species of Clusia that have photosynthetic patterns ranging from C3 photosynthesis to constitutive CAM. By linking changes in the expression of PEPC and PEPC kinase to day/night patterns of leaf gas exchange, organic acid, and soluble sugar contents under different environmental conditions, the genetic and metabolic limitations to CAM plasticity were assessed. The results indicate that PEPC expression is a major factor underpinning the genotypic capacity for CAM and that PEPC kinase expression does not appear to limit CAM. The day/night regulation of Ppck transcript abundance was found to be a consequence of CAM and the day/night cycling of associated metabolites, rather than the primary controlling factor for the temporal separation of carboxylation processes.

Degrees of crassulacean acid metabolism in tropical epiphytic and lithophytic ferns

Crassulacean acid metabolism (CAM) was observed in three species of tropical ferns, the epiphytes Microsorium punctatum and Polypodium crassifolium and the lithophyte Platycerium veitchii. Polypodium crassifolium and P. veitchii exhibited characteristics of weak CAM. Although no net nocturnal CO2 uptake was observed, the presence of CAM was inferred from nocturnal increases in titratable acidity of 4.7 and 4.1 µequiv (g fr wt)–1 respectively, a reduction in the rates of net CO2 evolution during the first half of the dark period, and the presence of a CAM-like decrease in net CO2 uptake during the early light period. In M. punctatum net CO2 uptake during the first half of the dark period was accompanied by an increase in titratable acidity of 39.2 µequiv (g fr wt)–1 and a pronounced reduction in net CO2 uptake during the early light period. When water was withheld from P. crassifolium and M. punctatum, net CO2 uptake during the light was reduced markedly but there was no change in the extent or patterns of CO2 exhange in the dark. As a consequence, the proportion of carbon gained due to CO2 fixation in the dark increased from 2.8 and 10% to 63.5 and 49.3%, respectively (100% being net CO2 uptake during the light plus the estimated CO2 uptake during the dark). After 9 days without added water, dark CO2 uptake was responsible for the maintenance of a net 24 h carbon gain in P. crassifolium. Platycerium veitchii, P. crassifolium and M. punctatum exhibited carbon isotope ratios of between –25.9 and –22.6‰ indicating that carbon isotope ratios may not, by themselves, be sufficient for the identification of weak CAM. We suggest that CAM may be more prevalent in tropical epiphytic and lithophytic ferns than currently envisaged.

Microplankton and its functional activity in zones of shallow hydrotherms in the Western Pacific

Water above the active underwater volcanoes on the slopes of Epi Island, New Hebrides, Raul Island, Kermadek Island and the 'Calypso' gas anomaly area. Bay of Plenty, New Zealand, being enriched with methane and hydrogen, contains abundant bacterioplankton with a biomass several times greater than the surrounding oceanic waters. Dark microbial CO2 uptake in these sites was 10-30 times greater than in adjacent oceanic waters. In the shallow underwater volcanic hydrotherm areas around Whale and White Islands (New Zealand), as well as in Matupi Harbour Bay (New Britain Island), significant rates of microbial chemosynthesis were recorded in the water column (5-10 mg C m3 day1). This was reflected in high microbial biomass: 20-60 mg C m~'. Over the field of algal-bacterial mats, it exceeded 100 mg C nv- The cell volume of bacteria growing in waters influ- enced by hydrothermal activity was 2-4 times greater than that of those growing in sea areas remote from hydrotherms. Primary phytoplankton production in these waters was significantly greater than in surrounding oceanic waters. A high activity of hydrogen- and methane-oxidizing bacteria, as well as thiobacilli, was also recorded there. The microzooplankton in areas of the sea influenced by hydrotherms (Whale Island) contained a large percentage of planktonic amoebae, up to 30 x 10-111. The probable ways in which shallow hydrothermal discharges influence the biota of coastal waters are discussed.

Discrimination Processes and Shifts in Carboxylation during the Phases of Crassulacean Acid Metabolism.

The magnitude and extent of Crassulacean acid metabolism (CAM) activity in two Clusia species was manipulated to investigate the regulation of the distinct CAM phases. First, in response to leaf-air vapor pressure deficit at night, changes in leaf conductance altered on-line carbon-isotope discrimination throughout the theoretical range for dark CO2 uptake during CAM. These ranged from the limit set by phosphoenolpyruvate carboxylase (PEPc) (-6[per mille (thousand) sign], [delta]13C equivalent of -2[per mille (thousand) sign]) to that imposed by diffusion limitation (+4[per mille (thousand) sign], [delta]13C equivalent of -12[per mille (thousand) sign]), but the lowest carbon-isotope discrimination occurred when P[square root]pa was only 0.7. Second, when the availability of external or internal sources of CO2 was reduced for both field- and greenhouse-grown plants, CO2 uptake by day via PEPc during phase II largely compensated. Third, by reducing the dark period, plants accumulated low levels of acidity, and CO2 uptake occurred throughout the subsequent light period. Discrimination switched from being dominated by PEPc (phase II) to ribulose 1,5-bisphosphate carboxylase/oxygenase (phase III), with both enzymes active during phase IV. Under natural conditions, photochemical stability is maintained by extended PEPc activity in phase II, which enhances acid accumulation and delays decarboxylation until temperature and light stress are maximal at midday.

Photosynthesis of Littorella uniflora grown under two PAR regimes: C3 and CAM gas exchange and the regulation of internal CO2 and O2 concentrations.

The submersed aquatic macrophyte Littorella uniflora was grown under 50 and 300 μmol m-2 s-1 photosynthetically active radiation (PAR) (low and high PAR regimes) but identical sediment CO2 supply (1.0 mol m-3). The interactions between plant morphology, whole plant CO2 and O2 exchange, CAM activity, [CO2] i and [O2] i have been investigated in comparison with in vitro CO2 and PAR response characteristics (using 1 mm leaf sections). In terms of morphology, high-PAR-grown plants were smaller and leaves contained less chlorophyll, although root growth was proportionally larger. Gas exchange fluxes over roots and shoots of intact plants were similar in direction under the two PAR regimes, with the majority of CO2 uptake via the roots. Photosynthetic O2 evolution from intact plants was greater in high-PAR-grown L. uniflora (2.18 compared with 1.49 μmol O2g-1 fresh weight h-1 for the low PAR regime). Although net daytime CO2 uptake was similar for both PAR regimes (0.79 and 0.75 μmol g-1 fwt h-1), net dark CO2 uptake was at a higher rate (0.92 compared with 0.52 μmol CO2 g-1 fwt h-1), and dark fixation (as malic acid) was threefold greater in high PAR plants (ΔH+ 117 compared with 42 μmol H+ g-1 fwt). Comparison of dark CO2 uptake with dark fixation suggested that much of the CO2 fixed at night and regenerated during the day may be respiratory in origin (60% low PAR plants, 71% high PAR plants). Regeneration of CO2 from CAM could account for 62% of daytime CO2 supply in low PAR plants and 81% in high PAR plants. [CO2] i values (ranging from 0.42 to 1.03 mol m-3) were close to or above the concentration required to saturate photosynthesis in vitro (0.5 mol m-3) under both PAR regimes, and combined with the low [O2] i (2.6-4.3 mol m-3) should have suppressed photorespiration. However, PAR inside leaves would have been well below the in vitro light saturation requirement (850-1000 μmol m-2 s-1 for both treatments). Thus PAR rather than CO2 supply appeared to limit photosynthesis even in high PAR grown plants, and CAM appears to have an important role in the regulation of CO2 supply for photosynthesis in response to variation in light regime.

Photosynthetic acclimation in a terrestrial CAM bromeliad, Bromelia humilis Jacq.

Photosynthetic acclimation of a terrestrial CAM bromeliad -Bromelia humilis Jacq. to irradiance and nitrogen supply during growth was investigated under controlled conditions. Considerable acclimation to shade was realized through an increase in total chlorophyll, accessory pigments, an increase in chloroplast size and appressed to non-appressed thylakoid membrane ratio, and a reduction in chlorophyll a/b ratio. A relatively high apparent photon yield in shade was associated with a low light-compensation point and low dark-respiration rate. Light saturation of integrated dark CO2 uptake occurred at a relatively low total daily photosynthetic photon flux density (PPFD) and was not influenced by growth PPFD level. Maximum photosynthetic capacity expressed per unit chlorophyll and nitrogen use efficiency were significantly higher in high-PPFD-grown plants. In addition, apparent photon yield was only slightly reduced in high-PPFD-grown plants with nitrogen, while maximum photosynthetic rate remained the same indicating radiationless dissipation of excess light. Very high levels of recycling of respiratory carbon were observed under nitrogen deficiency in plants grown in both high and low PPFD. The extensive photosynthetic acclimation of B. humilis is discussed in terms of contrasting exposure and limitations of its natural environment.

AMMONIUM, NITRATE, PHOSPHATE, AND INORGANIC CARBON UPTAKE IN AN OLIGOTROPHIC LAKE: SEASONAL VARIATIONS AMONG LIGHT RESPONSE VARIABLES1

ABSTRACTThe dependence of substrate saturated uptake of 15NH4+, 15NO3−, 32PO43−, and 14CO2 on photosynthetic photon flux density (PPFD or photsynthetically active radiation, 400–700 nm) was characterized seasonally in oligotrophic Flathead Lake, Montana. PO43− uptake was not dependent upon PPFD at any time of the year, whereas NH4+, NO3−, and CO2 uptake were consistently dependent on PPFD over all seasons. Maximal rates of NH4+, NO3− and CO2 uptake usually occurred near 40% of surface PPFD, which corresponded to about 5 m in the lake; inhibition was evident at PPFD levels greater than 40%. NH4+, NO3− and PO43− were incorporated in the dark at measurable rates most of the year, whereas dark CO2 uptake was always near 0 relative to light uptake. CO2 and NO3− uptake were more strongly influenced by PPFD than was NH43− uptake. The PPFD dependence of PO43−, NH4+, NO3− and CO2 uptake may affect algal growth and nutrient status by influencing the balance in diel and seasonal C:N:P uptake ratios.

The Regulation of Citric Acid Accumulation and Carbon Recycling During CAM inAnanas comosus

The carbon balance of shade-grown Ananas comosus was investigated with regard to nitrogen supply and responses to high PAR. Net dark CO2 uptake was reduced from 61.2 to 38.5 mmol CO2 m−2 in N limited (−N) plants grown under low PAR (60 μmol m−2 s−1) and apparent photon yield declined from 0.066 to 0.034 (mol 02.mol−1 photon), although photosynthetic capacities (measured under 5% CO2) were similar. Following transfer for 7 d to high PAR (600. μmol m−2 s−1), net CO2 uptake at night increased by 14% in +N plants, and daytime photosynthetic capacity was higher, with a maximum value of 7.8 μmol m−2 s−1. The magnitude of dark CO2 fixation during CAM was measured as dawn—dusk variations in leaf-sap titratable acidity (ΔH+) and as the proportion of malic and citric acids. The contribution from re-fixation of respiratory CO2 recycling (measured as the difference between net CO2 uptake and malic acid accumulation) varied with growth conditions, although it was generally lower (30%) than reported for other bromeliads. Assuming a stoichiometry of 2H+: malate and 3H+: citrate, there was a good agreement between titratable protons and enzymatically determined organic acids. The accumulation of citric acid was related to nitrogen supply and PAR regime, increasing from 7.0 mol m−3 (+N plants) to 18 mol m−3 (−N plants) when plants were transferred to high PAR; malate: citrate ratios decreased from 13.1 to 2.5 under these conditions. Under the low PAR regime, leaf-sap osmotic pressure increased at night in proportion to malic acid accumulation. However, following the transfer to high PAR for 7 d, there was a much greater depletion of soluble sugars at night which corresponded to a decrease in leaf-sap osmotic pressure. Although a role for citric acid in CAM has not been properly defined, it appears that the accepted stoichiometry for CAM in terms of gas exchange, titratable acidity, malic acid and osmotic pressure may not hold for plants which accumulate citric acid.

Carbon balance during CAM: an assessment of respiratory CO2 recycling in the epiphytic bromeliads Aechmea nudicaulis and Aechmea fendleri

Abstract The regulation of crassulacean acid metabolism (CAM) under controlled environmental conditions has been investigated for two tropical epiphytes, relating plant water and carbon balance to growth form and habitat preference under natural conditions. Aechmea fendleri is restricted to wet, upper montane regions of Trinidad, while A. nudicaulis has a wider distribution extending into more arid regions of the island. Morphological characteristics of these plants are related to habitat preference in terms of leaf succulence (0.44 and 0.94 kg m−2 for the two species respectively) and a distinct layer of water storage parenchyma in A. nudicaulis In contrast, the thinner leaves of A. fendleri contain little water‐storage parenchyma and less chlorenchyma per unit area, but the plants have a more open leaf rosette. The two species differ in expression of CAM, since the proportion of respiratory CO2 recycled as part of CAM had been found to be much lower in A. fendleri This study compared the efficiency of water use and role of respiratory CO2 recycling under two PAR regimes (300 and 120 μnol m−2 s−1) and three night temperatures (12, 18 and 25 °C). Dark CO2 uptake rates for both species were comparable to plants in the field (maximum of 2.3 ± 0.2 μmol m−2s−1± SD, n= 3). Total net CO2 uptake at night increased on leaf area basis with temperature for both species under higher PAR, although under the low PAR regime CO2 uptake was maximal at 18 °C. Water‐use efficiency (WUE) increased at 18 °C and 25 °C during dark CO2 uptake (Phase I) and also during late afternoon photosynthesis (Phase IV) in both species. For A. fendleri, dawn to dusk changes in titrable acidity (ΔH +) were similar under high and low PAR, although ΔH+ was correlated to night temperature and PAR in A. nudicaulis. The proportion of ΔH+ derived from respiratory CO2 also varied with experimental conditions. Thus percentage recycling was lower in A. fendleri under high PAR (0–10%), but was only reduced at 18 °C under low PAR. Recycling by A. nudicaulis ranged from 32–42% under high PAR, but was also reduced to 6% under low PAR at 18 °C; at 12 °C and 25 °C, recycling was 37% and 52% respectively. Previous studies have suggested a relationship between the proportion of recycling and degree of water stress. This study indicated that CAM as a CO2 concentrating mechanism regulates both water‐use efficiency and plant carbon balance in these epiphytes, in response to PAR and night temperature. However, the precise relationship between respiratory processes and the balance between external and internal sources of CO2 is as yet unresolved.

Comparative ecophysiology of CAM and C3 bromeliads. III. Environmental influences on CO2 assimilation and transpiration

Abstract Field measurements of the gas exchange of epiphytic bromeliads were made during the dry season in Trinidad in order to compare carbon assimilation with water use in CAM and C3 photosynthesis.The expression of CAM was found to be directly influenced by habitat and microclimate. The timing of nocturnal CO2 uptake was restricted to the end of the dark period in plants found at drier habitats, and stomatal conductance in two CAM species was found to respond directly to humidity or temperature. Total night‐time CO2 uptake, when compared with malic‐acid formation (measured as the dawn‐dusk difference in acidity, ΔH+), could only account for 10–40% of the total ΔH+ accumulated. The remaining malic acid must have been derived from the refixation of respired CO2 (recycling). Within the genus Aechmea (12 samples from four species), recycling was significantly correlated with night temperature at the six sample sites. Recycling was lowest in A. fendleri (54% of ΔH+ derived from respired CO2), a CAM bromeliad with little water‐storage parenchyma that is restricted to wetter, cooler regions of Trinidad.Gas‐exchange rates of C3 bromeliads were found to be similar to those of the CAM bromeliads, with CO2 uptake from 1 to 3 μmol m−2 s−1 and stomatal conductances generally up to 100 mmol m−2 s−1. The midday depression of photosynthesis occurred in exposed habitats, although photosynthetically active radiation (PAR) limited photosynthesis in shaded habitats. CO2 uptake of the C3 bromeliad Guzmania lingulata was saturated at around 500 μmol m−2 s−1 PAR, suggesting that epiphytic plants found in the shaded forest understorey are shade‐tolerant rather than shade‐demanding.Transpiration ratios (TR) during CO2 fixation in CAM (Phase I and IV) and C3 bromeliads were compared at different sites in order to assess the efficiency of water utilization. For the epiphytes displaying marked uptake of CO2, TR were found to be lower than many previously published values. In addition, the average TR values were very similar for dark CO2 uptake in CAM (42 ± 41, n= 12), Phase IV of CAM (69 ± 36, n= 3) and for C3 photosynthesis (99 ± 73, n= 4) in these plants. It appears that recycling of respired CO2 by CAM bromeliads and efficient use of water in all phases of CO2 uptake are physiological adaptations of bromeliads to arid microclimates in the humid tropics.

Dark CO2 uptake by the diatom Chaetoceros simplex in response to nitrogen pulsing

In a continuing investigation of dark CO2 uptake by nitrogen-limited cultures of the marine diatom Chaetoceros simplex (Bbsm), we expanded on several of our earlier conclusions regarding the potential application of this physiological response for measuring the degree and type of nitrogen limitation in phytoplankton populations. First, the duration over which the maximal enhancement of dark 14CO2 uptake was sustained after NH4+enrichment was a function both of the concentration of added NH4+and the standing crop of phytoplankton nitrogen — in effect, the total N demand. Second, pulsing with NH4+for a given degree of N-limitation always produced the same level of enhanced dark CO2 uptake regardless of whether the cultures were preconditioned with oxidized or reduced nitrogen. In contrast, urea pulsing led to reduced dark CO2 uptake, but the effect was most pronounced in cells grown on NO3−. And third, the assay could be used to distinguish readily between no, moderate, and severe N limitation. The degree of severe N limitation was quantitatively correlated with the degree of enhanced dark CO2 uptake, but this relationship was not so clear in the region of moderate N limitation. The main advantage of the assay is that it is a relatively simple and effective alternative to more complicated techniques for gauging the degree and form of N limitation in phytoplankton. Further evaluation will be required, both in the laboratory and field, before the assay can be calibrated for quantitative use.

Measurements of Diel Rates of Bacterial Secondary Production in Aquatic Environments

Measurements of bacterial secondary production were carried out during 13 diel studies at one coastal marine station and in five lakes differing with respect to nutrient concentration and primary production. Bacterial secondary production was measured in situ every 3 to 5 h by [H]thymidine incorporation into DNA. In some of the diel studies, these results were compared with results obtained from dark CO(2) uptake and frequency of dividing cells. Only minor diel changes were observed. The rate of [H]thymidine incorporation into DNA and the frequency of dividing cells varied from 23 to 194% of the diel mean. The dark CO(2) uptake rate varied from 12 to 259% of the diel mean. An analysis of variance demonstrated that no specific time periods during 24 h showed significantly different production rates, supporting the idea that bacterial activities in natural assemblages are controlled by a variety of events. The best correction (r = 0.74) was obtained between the [H]thymidine incorporation and frequency of dividing cells procedures from the lake water samples. The actual production rates calculated by [H]thymidine incorporation into DNA were appreciably lower than those obtained by the frequency of dividing cells and the dark CO(2) uptake techniques. Diel rates of bacterial production are discussed in relation to sampling frequency, statistical errors, and choice of method.

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

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.

Crassulacean acid metabolism in the seasonally submerged aquatic Isoetes howellii.

Evidence to date is consistent with the hypothesis that the submerged aquatic Isoetes howellii Engelmann possesses crassulacean acid metabolism. Quantitative 14C uptake studies indicate that CO2 assimilation in both the light and dark are functions of pH and total inorganic carbon level. In both the light and dark, maximum uptake rates in 0.6 mM NaHCO3 were double the rates in 0.3 mM NaHCO3. At both carbon levels there was a large drop in carbon assimilation rate between pH 6 and 8. In nature water pH and inorganic carbon level fluctuated diurnally thus complicating the determination of the contribution of light vs dark CO2 uptake to the total carbon gain. On a sunny day between 0600 and 1200 h water chemistry changed markedly with ∼40% reduction in total carbon, ∼2 pH unit rise resulting from ∼100% depletion of free CO2. Under such conditions daytime deacidification in Isoetes leaves was 88% complete by noon. In contrast, on an overcast day, reduction of carbon in the water was much slower, deacidification was only 46% complete by noon and substantial malic acid levels remained in the leaves at the end of the day. Upon emergence crassulacean acid metabolism was largely lost in Isoetes leaves. Preliminary estimates suggest that under natural submerged conditions, early morning photosynthetic rates may be substantially higher than dark CO2 uptake rates, though uptake rates throughout much of the day could be substantially lower than nightime CO2 assimilation.