Environmental regulation of carbon isotope composition and crassulacean acid metabolism in three plant communities along a water availability gradient

Expression of crassulacean acid metabolism (CAM) is characterized by extreme variability within and between taxa and its sensitivity to environmental variation. In this study, we determined seasonal fluctuations in CAM photosynthesis with measurements of nocturnal tissue acidification and carbon isotopic composition (δ¹³C) of bulk tissue and extracted sugars in three plant communities along a precipitation gradient (500, 700, and 1,000 mm year⁻¹) on the Yucatan Peninsula. We also related the degree of CAM to light habitat and relative abundance of species in the three sites. For all species, the greatest tissue acid accumulation occurred during the rainy season. In the 500 mm site, tissue acidification was greater for the species growing at 30% of daily total photon flux density (PFD) than species growing at 80% PFD. Whereas in the two wetter sites, the species growing at 80% total PFD had greater tissue acidification. All species had values of bulk tissue δ¹³C less negative than −20‰, indicating strong CAM activity. The bulk tissue δ¹³C values in plants from the 500 mm site were 2‰ less negative than in plants from the wetter sites, and the only species growing in the three communities, Acanthocereus tetragonus (Cactaceae), showed a significant negative relationship between both bulk tissue and sugar δ¹³C values and annual rainfall, consistent with greater CO₂ assimilation through the CAM pathway with decreasing water availability. Overall, variation in the use of CAM photosynthesis was related to water and light availability and CAM appeared to be more ecologically important in the tropical dry forests than in the coastal dune.     

A malic Acid permease in isolated vacuoles of a crassulacean Acid metabolism plant

Pine (Pinus silvestris L.) trees subjected to relatively low concentration of SO₂ in the field emit H₂S from the needles, as demonstrated by gas chromatographic analysis after preconcentration on a molecular sieve. H₂S is the only reduced sulfurous compound emitted from SO₂ fumigated leaves. The emission is light and SO₂ concentration dependent. Pine trees in the field and in laboratory experiments continue to emit H₂S several hours after the termination of prolonged SO₂ fumigation. The maximum emission rates observed from pine trees in the field and in laboratory experiments, 14 and 20 nanomoles per milligram chlorophyll per hour respectively, are about the activity expected for the sulfur assimilation pathway in the chloroplasts.     

Hydrogen, oxygen, and carbon isotope ratios of cellulose from submerged aquatic crassulacean Acid metabolism and non-crassulacean Acid metabolism plants

Isotope ratios of cellulose and cellulose nitrate from aquatic Crassulacean acid metabolism (CAM) and non-CAM plants were determined. Cellulose oxygen istope ratios for all plants that grew together were virtually identical, whereas large differences were observed for hydrogen isotope ratios of cellulose nitrate between CAM and non-CAM plants. Carbon isotope ratios of cellulose nitrate did not differentiate CAM from non-CAM plants.     

Maintenance carbon cycle in crassulacean Acid metabolism plant leaves : source and compartmentation of carbon for nocturnal malate synthesis

The reciprocal relationship between diurnal changes in organic acid and storage carbohydrate was examined in the leaves of three Crassulacean acid metabolism plants. It was found that depletion of leaf hexoses at night was sufficient to account quantitatively for increase in malate in Ananas comosus but not in Sedum telephium or Kalanchoë daigremontiana. Fructose and to a lesser extent glucose underwent the largest changes. Glucose levels in S. telephium leaves oscillated diurnally but were not reciprocally related to malate fluctuations.

Analysis of isolated protoplasts and vacuoles from leaves of A. comosus and S. telephium revealed that vacuoles contain a large percentage (>50%) of the protoplast glucose, fructose and malate, citrate, isocitrate, ascorbate and succinate. Sucrose, a major constituent of intact leaves, was not detectable or was at extremely low levels in protoplasts and vacuoles from both plants.

In isolated vacuoles from both A. comosus and S. telephium, hexose levels decreased at night at the same time malate increased. Only in A. comosus, however, could hexose metabolism account for a significant amount of the nocturnal increase in malate. We conclude that, in A. comosus, soluble sugars are part of the daily maintenance carbon cycle and that the vacuole plays a dynamic role in the diurnal carbon assimilation cycle of this Crassulacean acid metabolism plant.

     

植物呼吸释放CO2碳同位素变化研究进展

稳定性碳同位素是研究碳循环的有效手段。植物呼吸释放CO_2的碳同位素(δ~(13)C_R)变化是研究植物或生态系统与大气碳交换的重要方法,并可以揭示植物的生理过程、碳分配方式及其对环境变化的响应。介绍了目前国内外关于植物δ~(13)C_R变化的研究概况,植物不同器官δ~(13)C_R值及其日变化幅度趋势一致:叶片根系树干/茎,不同功能群植物其呼吸释放CO_2碳同位素组成存在差异。但植物δ~(13)C_R值日变化与呼吸底物的相关性在不同的研究中结果并不一致。导致植物呼吸δ~(13)C_R发生变化的主要原因为光合同位素效应、呼吸底物的供给及呼吸代谢中间产物利用、碳代谢相关酶的活性、LEDR(light enhanced dark respiration)、植物的遗传特性及外部环境改变。目前国际上已有较多关于导致植物呼吸δ~(13)C_R发生变化原因的研究,但内在机制的研究尚未完善。该领域研究在国内鲜有报道,因此,亟需加强我国关于植物δ~(13)C_R短期变化及其潜在呼吸代谢机制的研究。     

Carbon isotope ratios in crassulacean Acid metabolism plants: seasonal patterns from plants in natural stands

A year round study of photosynthesis and carbon isotope fractionation was conducted with plants of Opuntia phaeacantha Engelm. and Yucca baccata Torr. occurring in natural stands at elevations of 525, 970, 1450 and 1900 m. Plant water potentials and the daytime pattern of ¹⁴CO₂ photosynthesis were similar for all cacti along the elevational gradient, despite significant differences in temperature regime and soil water status. Carbon isotope ratios of total tissue and soluble extract fractions were relatively constant throughtout the entire year. Additionally, the σ¹³C values were similar in all plants of the same species along the elevational gradient, i.e. −12.5 ± 0.86 ‰ for O. phaeacantha and −15.7 ± 0.95 ‰ for Y. baccata. The results of this study indicate Crassulacean acid metabolism predominates as the major carbon pathway of these plants, which do not facultatively utilize the reductive pentose phosphate cycle of photosynthesis as the primary carboxylation reaction.     

Day-to-night variations of cytoplasmic pH in a crassulacean acid metabolism plant

Summary In crassulacean acid metabolism (CAM) large amounts of malic acid are redistributed between vacuole and cytoplasm in the course of night-to-day transitions. The corresponding changes of the cytoplasmic pH (pH_cyt) were monitored in mesophyll protoplasts from the CAM plantKalanchoe daigremontiana Hamet et Perrier by ratiometric fluorimetry with the fluorescent dye 2′,7′-bis-(2-carboxyethyl)-5-(and-6-)carboxyfluorescein as a pH_cyt indicator. At the beginning of the light phase, pH_cyt was slightly alkaline (about 7.5). It dropped during midday by about 0.3 pH units before recovering again in the late-day-to-early-dark phase. In the physiological context the variation in pH_cyt may be a component of CAM regulation. Due to its pH sensitivity, phosphoenolpyruvate carboxylase appears as a likely target enzyme. From monitoring ΔpH_cyt in response to loading the cytoplasm with the weak acid salt K-acetate a cytoplasmic H⁺-buffer capacity in the order of 65 mM H+ per pH unit was estimated at a pH_cyt of about 7.5. With this value, an acid load of the cytoplasm by about 10 mM malic acid can be estimated as the cause of the observed drop in pH_cyt. A diurnal oscillation in pH_cyt and a quantitatively similar cytoplasmic malic acid is predicted from an established mathematical model which allows simulation of the CAM dynamics. The similarity of model predictions and experimental data supports the view put forward in this model that a phase transition of the tonoplast is an essential functional element in CAM dynamics.     

Phosphoenolpyruvate carboxykinase in plants exhibiting crassulacean Acid metabolism

Phosphoenolpyruvate carboxykinase has been found in significant activities in a number of plants exhibiting Crassulacean acid metabolism. Thirty-five species were surveyed for phosphoenolpyruvate carboxykinase, phosphoenolpyruvate carboxylase, ribulose diphosphate carboxylase, malic enzyme, and malate dehydrogenase (NAD). Plants which showed high activities of malic enzyme contained no detectable phosphoenolpyruvate carboxykinase, while plants with high activities of the latter enzyme contained little malic enzyme. It is proposed that phosphoenolpyruvate carboxykinase acts as a decarboxylase during the light period, furnishing CO₂ for the pentose cycle and phosphoenolpyruvate for gluconeogenesis.     

Temperature features of enzymes affecting crassulacean Acid metabolism

Enzymes involved in malic acid production via a pathway with 2 carboxylation reactions and in malic acid conversion via total oxidation have been demonstrated in mitochondria of Bryophyllum tubiflorum Harv. Activation of the mitochondria by Tween 40 was necessary to reveal part of the enzyme activities. The temperature behavior of the enzymes has been investigated, revealing optimal activity of acid-producing enzymes at 35°. Even at 53° the optimum for acid-converting enzymes was not yet reached. From the simultaneous action of acid-producing and acid-converting enzyme systems the overall result at different temperatures was established. Up to 15° the net result was a malic acid production. Moderate temperatures brought about a decrease in this accumulation, which was partly accompanied by a shift to isocitrate production, while at higher temperatures total oxidation of the acids exceeded the production.     

陆生植物稳定碳同位素组成与全球变化

分析了大气CO₂浓度、温度、降水和海拔高度等环境因素对陆生植物稳定性碳同位素组分的影响及其作用机理,综述了国内外碳稳定同位素技术在全球变化研究中的进展和应用,如重建大气CO₂浓度变化,揭示温度、降水对树木生长的“滞后效应”和“幼龄效应”,确定不同光合型植物随海拔高度的分布变化,以及通过碳稳定同位素技术揭示不同时间尺度上和不同气候条件下的植物水分利用效率变化及不同生活型植物的水分利用效率差异,并探讨研究中存在的问题及其研究前景.     

Variation in carbon isotope composition among years in the riparian tree Populus fremontii

Water availability is an important factor limiting the productivity of desert plants but little is known about the impact of water-limiting conditions on the physiology of plants in mesic environments. Riparian ecosystems of the western US receive significantly more water than the surrounding desert environments but experience dramatic interannual fluctuations in water availability because both stream flow and precipitation are highly variable over time. This variability results in different growing conditions each year which may influence the physiology of riparian species such as Fremont cottonwood (Populus fremontii), the dominant, native canopy tree species in lowland southwestern US river systems. We wished to determine if the physiology of this species varies among years, what climatic parameters are related to any observed physiological variation and if individuals within a P. fremontii population differ in their physiological response to variation through time. We collected tree ring cores from a central New Mexico cottonwood population and analyzed carbon isotope composition (δ¹³C) in each year from 1981 to 1995. We used δ¹³C analysis in this study because it allowed us to obtain multi-year estimates of physiological activity. During these years, mean stream flow at our study site ranged over two orders of magnitude from 0.82 to 80.94 m³ s⁻¹, precipitation ranged fourfold from 49 to 215 ccmm and mean temperature ranged from 20.5 to 22.6°C during the growing season. δ¹³C varied from a low of −26.7‰ in 1984 to a high of −24.7‰ in 1981. Low δ¹³C values were associated with years in which stream flow and/or precipitation were high and temperature was low. The opposite was true of years with high δ¹³C values. We observed a strong linear relationship between δ¹³C and stream flow during years when stream flow was <25 m³ s⁻¹ but no significant relationship between these variables when stream flow was >25 m³ s⁻¹. Additionally, there was a linear relationship between δ¹³C and precipitation during years when stream flow was <25 m³ s⁻¹ but not in years when stream flow was >25 m³ s⁻¹. These data suggest that above a threshold of total stream flow, increased flow does not influence physiology. Below this threshold, precipitation can be an important water source. The ten individuals within our study population varied significantly in mean δ¹³C values but responded to interannual variation in a similar manner (i.e., all individuals had low δ¹³C values when water was abundant). These results suggest that precipitation as well as stream flow are important factors influencing the physiology of this riparian tree. Received: 25 November 1998 / Accepted: 9 February 1999     

Photosynthetic studies with mesophyll protoplasts from Notonia grandiflora, a crassulacean acid metabolism plant

A method is described for rapid enzymatic isolation of mesophyll protoplasts and cells from the crassulacean acid metabolism (CAM) plant Notonia grandiflora DC. The mesophyll protoplasts exhibited high rates of ¹⁴CO₂ fixation both in the light (45 μmol of CO₂ fixed mg^?1 Chl h^?1) and in the dark (20 μmol of CO₂ fixed mg^?1 Chl h^?1). The protoplasts also showed O₂ evolution (40 μmol of O₂ evolved mg^?1 Chl h^?1) without added bicarbonate. Exogenously added bicarbonate had no stimulating effect on the O₂ evolution. Analyses of early photosynthetic products in the light showed the formation of both C₃ and C₄ acids. Aspartate was found to be a predominant photosynthate.     

Carbon isotope composition of biochemical fractions isolated from leaves of Bryophyllum daigremontianum berger,a plant with crassulacean acid metabolism: Some physiological aspects related to CO2 dark fixation

Isotope analysis of the biochemical fractions isolated quantitatively from young and mature leaves of Bryophyllum daigremontianum Berger have been carried out before and after a dark period of accumulation of organic acids. The mature leaf is enriched in ¹³C compared to the young leaf. The ¹³C values of the different leaf constituents vary between the ¹³C values of C₄ plants (-11) and those of C₃ plants (-27). During the dark period, the two types of leaves store organic acids with ¹³C values of -15 and lose insoluble sugars, including starch with a ¹³C value of -12. Furthermore, young leaves store phosphorylated compounds with ¹³C values of -11 and lose weakly polymerised sugars with ¹³C values of -18. These results led to the formulation of a hypothesis of the origin of the two substrates of -carboxylation: phosphoenolpyruvate arises from the glycolytic breakdown of the insoluble sugars rich in ¹³C, and the major portion of the CO₂ is the result of the complete breakdown (respiration) of the soluble sugars rich in ¹²C. The existence of two independent sugar pools leads to the assumption that there are two separate glycolytic pathways. The ¹³C enrichment of the stored products of the young leaves in the day seems to be the result of a weak discrimination for ¹³C by ribulose diphosphate carboxylase, which reassimilates to a great extent the CO₂ released from malate accumulated in the night.Abbreviations CAM crassulacean acid metabolism - C₃ metabolism metabolism with primary carbon fixed by the Calvin and Benson pathway - C₄ metabolism metabolism with primary carbon fixed by the Hatch and Slack pathway - ¹³C() (R_sample-R_PDB) 10³/R_PDB where PDB=Pee Dee belemnite (belemite from the Pee Dee formation South Carolina) and R=¹³C/¹²C - NAD-MDH(EC1.1.1.37) NAD-malate dehydrogenase - NADP-ME (EC1.1.1.40) NADP-malic enzyme - PEP phosphoenolpyruvate - PEPC (EC4.1.1.31) PEP carboxylase - PGA phosphoglyceric acid - Py.di-PK(EC2.7.9.1) pyruvate, Pi-dikinase - RuDP ribulose diphosphate - RuDPC (EC4.1.1.39) RuDP carboxylase     

C/C ratio changes in crassulacean Acid metabolism plants

¹³C/¹²C ratios have been found in totally combusted leaves of Crassulacean acid metabolism plants to range from −14 to −33 δ ¹³C‰ compared with a limestone standard. Crassulacean acid metabolism plants apparently utilize both ribulose-1, 5-diphosphate carboxylase and phosphoenolpyruvate carboxylase to assimilate atmospheric CO₂ and, depending on environmental conditions, have ¹³C/¹²C ratios indicative of either carboxylase or to any intermediate value. The degree of discrimination against ¹³C and the resultant ¹³C/¹²C ratio from the photosynthetically fixed CO₂ is influenced by environmental conditions and is not a specific and fixed characteristic of a Crassulacean acid metabolism plant. Certain Crassulacean acid metabolism plants may shift their ratios as much as 17 δ ¹³C‰ in specific environments.     

Effects of competition on induction of crassulacean acid metabolism in a facultative CAM plant

Abiotic drivers of environmental stress have been found to induce CAM expression (nocturnal carboxylation) in facultative CAM species such as Mesembryanthemum crystallinum. The role played by biotic factors such as competition with non-CAM species in affecting CAM expression, however, remains largely understudied. This research investigated the effects of salt and water conditions on the competition between M. crystallinum and the C₃ grass Bromus mollis with which it is found to coexist in California’s coastal grasslands. We also investigated the extent to which CAM expression in M. crystallinum was affected by the intensity of the competition with B. mollis. We found that M. crystallinum had a competitive advantage over B. mollis in drought and saline conditions, while B. mollis exerted strong competitive effects on M. crystallinum in access to light and soil nutrients in high water conditions. This strong competitive effect even outweighed the favorable effects of salt or water additions in increasing the biomass and productivity of M. crystallinum in mixture. Regardless of salt conditions, M. crystallinum did not switch to CAM photosynthesis in response to this strong competitive effect from B. mollis. Disturbance (i.e., grass cutting) reduced the competitive pressure by B. mollis and allowed for CAM expression in M. crystallinum when it was grown mixed with B. mollis. We suggest that moderate competition with other functional groups can enhance CAM expression in M. crystallinum, thereby affecting its plasticity and ability to cope with biological stress.     

Variation in the carbon isotope composition of Semaprochilodus insignis, a detritivorous fish associated with oligotrophic and eutrophic Amazonian rivers

The Amazonian freshwater fish, Semaprochilodus insignis , varies in carbon isotopic composition during residence in different habitats within its home range. The results indicate that carbon produced in black-water systems contributes to fish stocks harvested from white-water systems, and S. insignis populations in the Central Amazonia are more integrated than previously thought.     

Carbon isotope composition of intermediates of the starch-malate sequence and level of the crassulacean acid metabolism in leaves of Kalanchoe blossfeldiana Tom Thumb

Isotype analyses were performed on biochemical fractions isolated from leaves of Kalanchoe blossfeldiana Tom Thumb. during aging under long days or short days. Irrespective of the age or photoperiodic conditions, the intermediates of the starch-malate sequence (starch, phosphorylated compounds and organic acids) have a level of ¹³C higher than that of soluble sugars, cellulose and hemicellulose. In short days, the activity of the crassulacean acid metabolism pathway is predominant as compared to that of C₃ pathway: leaves accumulate organic acids, rich in ¹³C. In long days, the activity of the crassulacean acid metabolism pathway increases as the leaves age, remaining, however, relatively low as compared to that of C₃ pathway: leaves accumulate soluble sugars, poor in ¹³C. After photoperiodic change (long daysshort days), isotopic modifications of starch and organic acids suggest evidence for a lag phase in the establishment of the crassulacean acid metabolism pathway specific to short days. The relative proportions of carbon from a C₃-origin (RuBPC acitivity as strong discriminating step, isotope discrimination in vivo=20) or C₄-origin (PEPC activity as weak discriminating step, isotope discrimination in vivo=4) present in the biochemical fractions were calculated from their ¹³C values. Under long days, 30 to 70% versus 80 to 100% under short days, of the carbon of the intermediates linked to the starch-malate sequence, or CAM pathway (starch, phosphorylated compounds and organic acids), have a C₄-origin. Products connected to the C₃ pathway (free sugars, cellulose, hemicellulose) have 0 to 50% of their carbon, arising from reuptake of the C₄ from malate, under long days versus 30 to 70% under short days.Abbreviations CAM crassulacean acid metabolism - CAM pathway pathway with malate accumulation by -carboxylation of PEP, arising from glycolysis of starch (starch-malate sequence) - C₃-metabolism metabolism with primary carbon fixed by the Calvin and Benson pathway (C₃-origin) - C₄-metabolism metabolism with primary carbon fixed by the Hatch and Slack pathway (C₄-origin) - C₃-pathway pathway with RuBPC activity and the Calvin and Benson pathway, irrespective of the CO₂-source, atmospheric or reuptake of the C₄ from malate - ¹³C()=(R_sample-R_PDR)10³/R_PDB where PDB Pee Dee belemnite (belemnite from the Pee Dee formation, South Carolina) and R=¹³C/¹²C - D isotope discrimination - PEP phosphoenolpyruvate - PEPC (EC 4.1.1.31) PEP carboxylase - PGA phosphoglyceric acid - Py.di-PK (EC 2.7.9.1) pyruvate, Pi-dikinase - RuBP ribulose bisphosphate - RuBPC (EC 4.1.1.39) RuBP carboxylase - SD short days - LD long days     

Malate decarboxylation in isolated mitochondria from the crassulacean acid metabolism plant Sedum praealtum

Mitochondria isolated from the Crassulacean acid metabolism plant Sedum praealtum were demonstrated to decarboxylate added malate at basal rates of 30–50 μmol mg^?1 original chlorophyll h^?1. The basal rate could be stimulated markedly by the addition of ADP, oxaloacetic acid, an uncoupler of oxidative phosphorylation, or NAD, with maximum rates of 70–100 μmol mg^?1 original chlorophyll h^?1 observed. These observed rates were high enough to account for a large proportion of the estimated rate of malate decarboxylation in vivo. The major products of malate oxidation by the mitochondria in most cases were found to be pyruvate and CO₂, indicating that malate oxidation in these mitochondria proceeds mainly through NAD malic enzyme rather than NAD malate dehydrogenase. Under conditions employed little of the pyruvate formed was further oxidized, suggesting a fate other than oxidation (conversion to starch) for this pyruvate. Malate decarboxylation by mitochondria and by partially purified NAD malic enzyme was markedly inhibited by NaHCO₃. A possible physiological role is suggested for this inhibition as a feedback control on the enzyme.