Effects of abscisic acid on CAM in Portulacaria afra

Water stress induces Crassulacean acid metabolism (CAM) in Portulacaria afra as manifested by day stomatal closure, organic acid fluctuation, and night CO₂ uptake. We now have evidence that abscisic acid treatment of leaves causes partial stomatal closure that is accompanied by the induction of CAM in a manner similar to water stress. There appears to be an inverse relationship between exogenous CO₂ uptake and decarboxylation of organic acids in that organic acids remain high during the day providing stomata are open. When stomata close, there is consumption of organic acids by decarboxylation. The hypothesis is that stomatal opening controls CAM in this species.This material is based upon work supported by the Science and Education Administration of the USDA under Competitive Grant No. 5901-0410-8-0018-0.     

Nocturnal water storage in plants having Crassulacean acid metabolism

Measurements of water uptake and transpiration, during the dark period of plants having Crassulacean acid metabolism (CAM) allow calculation of leaf-volume changes (V). Nocturnal leaf-volume changes of CAM plants have also been reported in the literature on the basis of waterdisplacement measurements. A third way of estimation is from measurements of turgor changes and cellular water-storage capacity using the pressure probe, cytomorphometry and the Scholander pressure chamber. An extension of the interpretation of results reported in the literature shows that for leaf succulent CAM plants the three different approaches give similar values of V ranging between 2.3 and 10.7% (v/v). It is evident that nocturnal malic-acid accumulation osmotically drives significant water storage in CAM leaf tissue.Abbreviations and symbols C_c water-storage capacity - E transporation (evaporational water loss) - P turgor pressure - U water uptake - V cell volume - cell-wall elastic modulus - osmotic pressure - CAM Crassulacean acid metabolism     

Crassulacean acid metabolism in selected terrestrial succulents in southeastern Jamaica,including two species in the Commelinaceae

Summary Using determinations of overnigh changes in tissue titratable acidity and of tissue stable carbon isotope ratios, 10 species of terrestrial succulents were investigatedin situ in southeastern Jamaica for the presence of Crassulacean acid metabolism (CAM). Eight of the 10 species exhibited CAM (sensu lato), confirming past reports of CAM inClusia flava (Clusiaceae),Bryophyllum pinnatum (Crassulaceae),Euphorbia tirucalli (Euphorbiaceae), andPedilanthus tithymaloides (Euphorbiaceae) and extending the number of species with CAM in two genera previously known to contain CAM species (Agave sobolifera [Agavaceae] andSansevieria metalllica [Liliaceae]). Stems of bothE. tirucalli andP. tithymaloides exhibited CAM while the leaves of both species were intermediate with regard to photosynthetic pathway. The lack of CAM acid fluctuations inTalinum paniculatum (Portulacaceae) was surprising given past findings with all other species investigated in this genus. Shoots ofPilea microphylla (Urticaceae) were C₃ yet were remarkable in their extremely high pH. Both species require further investigation. Nocturnal acid accumulations indicative of CAM were found inTripogandra multiflora andCallisia fragrans, both members of the Commelinaceae. This represents the first report of CAM (probably “CAM-cycling”) in this family.     

Stomatal responses to humidity and temperature in darkness

Stomatal responses to leaf temperature (T_l) and to the mole fractions of water vapour in the ambient air (w_a) and the leaf intercellular air spaces (w_i) were determined in darkness to remove the potential effects of changes in photosynthesis and intercellular CO₂ concentration. Both the steady‐state and kinetic responses of stomatal conductance (g_s) to w_a in darkness were found to be indistinguishable from those in the light. g_s showed a steep response to the difference (Δw) between w_a and w_i when w_a was varied. The response was much less steep when w_i was varied. Although stomatal apertures responded steeply to T_l when Δw was held constant at 17 mmol mol^?1, the response was much less steep when Δw was held constant at about zero. Similar results were obtained in the light for Δw = 15 mmol mol^?1 and Δw ≈ 0 mmol mol^?1. These results are discussed in the context of mechanisms for the stomatal response to humidity.     

Effects of growth regulators on the induction of Crassulacean acid metabolism in the facultative halophyte Mesembryanthemum crystallinum L.

The classical induction of Crassulacean acid metabolism (CAM) in Mesembryanthemum crystallinum L. by water stress is observed within one week when fourto five-week-old plants (grown under a 16/8 h photoperiod at ca. 600 mol quanta · m^–2 · s^–1) are irrigated with 350 mM NaCl. The induction of CAM was evaluated by measuring phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) and NADP-malic enzyme (NADP-ME, EC 4.1.1.82) activities and nocturnal increases in malate content and titratable acidity of leaf extracts, and the daily pattern of CO₂ exchange and stomatal conductance during the 7-d induction period. Three growth regulators, abscisic acid (ABA), farnesol (an antitranspirant and analog of ABA), and benzylaminopurine (BAP), were found to substitute for NaCl for induction of CAM when fed to plants in nutrient media. Daily irrigation with solutions containing micromolar levels (optimum ca. 10 micromolar) of these growth regulators led to the induction of CAM similar to that by high salt. Application of the growth regulators, like NaCl, caused large increases in the activity of NADP-ME and the activity and level of PEPCase, which are components of the biochemical machinery required for CAM. Western immunoblotting showed that the increased activity of PEPCase on addition of ABA, farnesol and BAP was mainly due to increased levels of the CAM-specific isoforms. Also, dehydration of cut leaves over 8.5 h under light resulted in a severalfold increase in PEPCase activity. An equivalent increase in PEPCase activity in excised leaves was also obtained by feeding 150 mM NaCl, or micromolar levels of ABA or BAP via the petiole, which supports results obtained by feeding the growth regulators to roots. However, the increase in PEPCase activity was inhibited by feeding high levels of BAP to cut leaves prior to dehydration, indicating a more complex response to the cytokinin. Abscisic acid may have a role in induction of CAM in M. crystallinum under natural conditions as there is previous evidence that induction by NaCl causes an increase in the content of ABA, but not cytokinins, in leaves of this species.Abbreviations ABA abscisic acid - BAP 6-benzylaminopurine - CAM Crassulacean acid metabolism - Chl chlorophyll - 2,4D 2,4-dichlorophenoxyacetic acid - NADP-ME NADP-malic enzyme - PEPCase phosphoenolpyruvate carboxylase Methyl jasmonate was generously provided by Dr. Vincent Franceschi (Botany Department, Washington State University). The anti-maize leaf PEPCase was kindly supplied by Dr. Tatsuo Sugiyama (Department of Agricultural Chemistry, Nagoya University, Japan) and the anti-Flaveria trinervia leaf PEPCase was kindly supplied by Dr. Samuel Sun (Department of Plant Molecular Physiology, University of Hawaii, Honulu). This work was funded in part by U.S. Department of Agriculture Competitive Grant 90-37280-5706 and an equipment grant (DMB 8515521) from the National Science Foundation. Ziyu Dai was supported in part by Guangxi Agricultural College and Ministry of Agriculture of the People's Republic of China     

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.     

Stomatal responses to humidity in isolated epidermes

The ability of guard cells to hydrate and dehydrate from the surrounding air was investigated using isolated epidermes of Tradescantia pallida and Vicia faba . Stomata were found to respond to the water vapour pressure on the outside and inside of the epidermis, but the response was more sensitive to the inside vapour pressure, and occurred in the presence or absence of living, turgid epidermal cells. Experiments using helium–oxygen air showed that guard cells hydrated and dehydrated entirely from water vapour, suggesting that there was no significant transfer of water from the epidermal tissue to the guard cells. The stomatal aperture achieved at any given vapour pressure was shown to be consistent with water potential equilibrium between the guard cells and the air near the bottom of the stomatal pore, and water vapour exchange through the external cuticle appeared to be unimportant for the responses. Although stomatal responses to humidity in isolated epidermes are the result of water potential equilibrium between the guard cells and the air near the bottom of the stomatal pore, stomatal responses to humidity in leaves are unlikely to be the result of a similar equilibrium.     

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     

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

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

Stomatal responses to jasmonic acid, linolenic acid and abscisic acid in wild-type and ABA-deficient tomato plants

Wild-type and abscisic acid (ABA) -deficient (sitiens) tomato plants were used to analyse the effects of abscisic acid (ABA), butyric acid (BA), jasmonic acid (JA) and linolenic acid (LA) on assimilation and transpiration rates in detached leaves taking up those substances into the transpiration stream. BA did not affect assimilation and transpiration rates. ABA decreased assimilation and transpiration in both wild-type and ABA-deficient mutants. JA reduced the assimilation rate in both lines but induced a significant reduction of transpiration in the wild type only. The response to LA in both lines was slower than that to JA.     

Responses to different external light rhythms by the circadian rhythm of Crassulacean acid metabolism in Kalanchoe daigremontiana

Time series of net CO₂ exchange ( J CO₂) and leaf conductivity for water vapour (gH₂O) were measured and subsequently analysed mathematically in the Crassulacean acid metabolism (CAM) plant Kalanchoe daigremontiana (Hamet et Perrier de la Bâthie) under constant environmental conditions and under imposed external rhythms of lower and higher light intensity. The time series were analysed by Fourier methods and a correlation analysis considering the first time derivatives of J CO₂, gH₂Oand photosynthetically active photon flux density (PPFD). The ratio of internal to external CO₂ (c_i/c_a) was also considered in the analysis, leading to a discussion of the interaction of stomata and carbon assimilation under periodic stimulation. It is suggested that for stimulation with frequencies close to the endogeneous circadian period, stomatal conductance and carbon assimilation oscillate synchronously, guard‐cell movements trailing behind changes in internal CO₂ with a delay of 10–15 min. For stimulation frequencies far shorter than the endogeneous period, this synchrony can be disturbed due to independent responses of stomata and assimilation to light pulses, leading to an arrhythmic gas exchange pattern. These results are discussed in the context of understanding circadian oscillations as the output of a multioscillator, multisignalling pathway system on the organismic and metabolic level.     

Stomatal responses in isolated epidermis of the crassulacean acid metabolism plant Kalanchoe daigremontiana Hamet et Perr.

The optimal conditions for opening of stomata in detached epidermis of the Crassulacean Acid Metabolism (CAM) plant Kalanchoe daigremontiana were determined. Stomatal opening in CO₂–free air was unaffected by light so subsequently all epidermal strips were incubated in the dark and in CO₂–free air. Apertures were maximal after 3 h incubation and were significantly greater at 15° C than 25° C. Thus stomata in isolated epidermis of this species can respond directly to temperature. Stomatal opening was greatest when the incubating buffer contained 17.6 mol m^–3 K⁺, but decreased linearly with increasing K⁺ concentrations between 17.6 and 300 mol m^–3; the decrease in aperture was shown to be associated with increasing osmotic potentials of the solutions. Reasons for this behaviour, which differs from that of many C₃ and C₄ species, are discussed. Stomatal apertures declined linearly upon incubation of epidermis on buffer solutions containing between 10^–11 and 10^–5 mol m^–3 abscisic acid (ABA). Hence stomata on isolated epidermis of K. daigremontiana respond to lower concentrations of ABA than those of any species reported previously.     

Opinion: Stomatal responses to light and CO2 depend on the mesophyll

The mechanisms by which stomata respond to red light and CO₂ are unknown, but much of the current literature assumes that these mechanisms reside wholly within the guard cells. However, responses of guard cells in isolated epidermes are typically much smaller than those in leaves, and there are several lines of evidence in the literature suggesting that the mesophyll is necessary for these responses in leaves. This paper advances the opinion that although guard cells may have small direct responses to red light and CO₂, most of the stomatal response to these factors in leaves is caused by an unknown signal that originates in the mesophyll.     

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     

The role of the mesophyll in stomatal responses to light and CO2

Stomatal responses to light and CO₂ were investigated using isolated epidermes of Tradescantia pallida , Vicia faba and Pisum sativum . Stomata in leaves of T. pallida and P. sativum responded to light and CO₂, but those from V. faba did not. Stomata in isolated epidermes of all three species could be opened on KCl solutions, but they showed no response to light or CO₂. However, when isolated epidermes of T. pallida and P. sativum were placed on an exposed mesophyll from a leaf of the same species or a different species, they regained responsiveness to light and CO₂. Stomatal responses in these epidermes were similar to those in leaves in that they responded rapidly and reversibly to changes in light and CO₂. Epidermes from V. faba did not respond to light or CO₂ when placed on mesophyll from any of the three species. Experiments with single optic fibres suggest that stomata were being regulated via signals from the mesophyll produced in response to light and CO₂ rather than being sensitized to light and CO₂ by the mesophyll. The data suggest that most of the stomatal response to CO₂ and light occurs in response to a signal generated by the mesophyll.     

Mass-spectrometric evidence for the double-carboxylation pathway of malate synthesis by Crassulacean acid metabolism plants in light

Phyllodia of the Crassulacean acid metabolism (CAM) plant Kalanchoë tubiflora were allowed to fix ¹³CO₂ in light and darkness during phase IV of the diurnal CAM cycle, and during prolongation of the regular light period. After ¹³CO₂ fixation in darkness, only singly labelled [¹³C]malate molecules were found. Fixation of ¹³CO₂ under illumination, however, produced singly labelled malate as well as malate molecules which carried label in two, three or four carbon atoms. When the irradiance during ¹³CO₂ fixation was increased, the proportion of singly labelled malate decreased in favour of plurally labelled malate. The irradiance, however, did not change either the ratio of labelled to unlabelled malate molecules found in the tissue after the ¹³CO₂ application, or the magnitude of malate accumulation during the treatment with label. The ability of the tissue to store malate and the labelling pattern changed throughout the duration of the prolonged light period. The results indicate that malate synthesis by CAM plants in light can proceed via a pathway containing two carboxylation steps, namely ribulose-1,5-bisphosphate-carboxylase/oxygenase (EC 4.1.1.39) and phosphoenolpyruvate carboxylase (EC 4.1.1.31) which operate in series and share common intermediates. It can be concluded that, in light, phosphoenolpyruvate carboxylase can also synthesize malate independently of the proceeding carboxylation step by ribulose-1,5-bisphosphate carboxylase/oxygenase.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase (EC 4.1.1.31) - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) - TMS trimethylsilyl     

Physiological responses of Opuntia ficus-indica to growth temperature

The influences of various day/night air temperatures on net CO₂ uptake and nocturnal acid accumulation were determined for Opuntia ficus-indica, complementing previous studies on the water relations and responses to photosynthetically active radiation (PAR) for this widely cultivated cactus. As for other Crassulacean acid metabolism (CAM) plants, net nocturnal CO₂ uptake had a relatively low optimal temperature, ranging from 11°C for plants grown at day/night air temperatures of 10°C/0°C to 23°C at 45°C/35°C. Stomatal opening, which occurred essentially only at night and was measured by changes in water vapor conductance, progressively decreased as the measurement temperature was raised. The CO₂ residual conductance, which describes chlorenchyma properties, had a temperature optimum a few degrees higher than the optimum for net CO₂ uptake at all growth temperatures. Nocturnal CO₂ uptake and acid accumulation summed over the whole night were maximal for growth temperatures near 25°C/15°C, CO₂ uptake decreasing more rapidly than acid accumulation as the growth temperature was raised. At day/night air temperatures that led to substantial nocturnal acid accumulation (25°C/15°C.). 90% saturation of acid accumulation required a higher total daily PAR than at non-optimal growth temperatures (10°C/0°C and 35°C/25°C). Also, the optimal temperature of net CO₂ uptake shifted downward when the plants were under drought conditions at all three growth temperatures tested, possibly reflecting an increased fractional importance of respiration at the higher temperatures during drought. Thus, water status, ambient PAR, and growth temperatures must all be considered when predicting the temperature response of gas exchange for O. ficus-indica and presumably for other CAM plants.