CO2 Exchange and Growth of the Crassulacean Acid Metabolism Plant Opuntia ficus-indica under Elevated CO2 in Open-Top Chambers

CO2 uptake, water vapor conductance, and biomass production of Opuntia ficus-indica, a Crassulacean acid metabolism species, were studied at CO2 concentrations of 370, 520, and 720 [mu]L L-1 in open-top chambers during a 23-week period. Nine weeks after planting, daily net CO2 uptake for basal cladodes at 520 and 720 [mu]L L-1 of CO2 was 76 and 98% higher, respectively, than at 370 [mu]L L-1. Eight weeks after daughter cladodes emerged, their daily net CO2 uptake was 35 and 49% higher at 520 and 720 [mu]L L-1 of C02, respectively, than at 370 [mu]L L-1. Daily water-use efficiency was 88% higher under elevated CO2 for basal cladodes and 57% higher for daughter cladodes. The daily net CO2 uptake capacity for basal cladodes increased for 4 weeks after planting and then remained fairly constant, whereas for daughter cladodes, it increased with cladode age, became maximal at 8 to 14 weeks, and then declined. The percentage enhancement in daily net CO2 uptake caused by elevated CO2 was greatest initially for basal cladodes and at 8 to 14 weeks for daughter cladodes. The chlorophyll content per unit fresh weight of chlorenchyma for daughter cladodes at 8 weeks was 19 and 62% lower in 520 and 720 [mu]L L-1 of CO2, respectively, compared with 370 [mu]L L-1. Despite the reduced chlorophyll content, plant biomass production during 23 weeks in 520 and 720 [mu]L L-1 of CO2 was 21 and 55% higher, respectively, than at 370 [mu]L L-1. The root dry weight nearly tripled as the C02 concentration was doubled, causing the root/shoot ratio to increase with CO2 concentration. During the 23-week period, elevated CO2 significantly increased CO2 uptake and biomass production of O. ficus-indica.     

Relationships between Photosynthetically Active Radiation, Nocturnal Acid Accumulation, and CO(2) Uptake for a Crassulacean Acid Metabolism Plant, Opuntia ficus-indica

The influences of photosynthetically active radiation (PAR) and water status on nocturnal Crassulacean acid metabolism (CAM) were quantitatively examined for a widely cultivated cactus, Opuntia ficus-indica (L.) Miller. When the total daily PAR was maintained at 10 moles photons per square meter per day but the instantaneous PAR level varied, the rate of nocturnal H⁺ accumulation (tissue acidification) became 90% saturated near 700 micromoles per square meter per second, a PAR level typical for similar light saturation of C₃ photosynthesis. The total nocturnal H⁺ accumulation and CO₂ uptake reached 90% of maximum for a total daily PAR of about 22 moles per square meter per day. Light compensation occurred near 0 moles per square meter per day for nocturnal H⁺ accumulation and 4 moles per square meter per day for CO₂ uptake. Above a total daily PAR of 36 moles per square meter per day or for an instantaneous PAR of 1150 micromoles per square meter per second for more than 6 hours, the nocturnal H⁺ accumulation actually decreased. This inhibition, which occurred at PAR levels just above those occurring in the field, was accompanied by a substantial decrease in chlorophyll content over a 1-week period.

A minimum ratio of H⁺ accumulated to CO₂ taken up of 2.5 averaged over the night occurred for a total daily PAR of 31 moles per square meter per day under wet conditions. About 2 to 6 hours into the night under such conditions, a minimum H⁺-to-CO₂ ratio of 2.0 was observed. Under progressively drier conditions, both nocturnal H⁺ accumulation and CO₂ uptake decreased, but the H⁺-to-CO₂ ratio increased. A ratio of two H⁺ per CO₂ is consistent with the H⁺ production accompanying the conversion of starch to malic acid, and it apparently occurs for O. ficus-indica when CAM CO₂ uptake is strongly favored over respiratory activity.

     

Drought Adaptation in Opuntia basilaris: Significance of Recycling Carbon through Crassulacean Acid Metabolism

Contrasting metabolic regimes operate in Opuntia basilaris Engelm. and Bigelov, before and after precipitation. During periods of drought, atmospheric CO₂ exchange and transpiration are greatly reduced throughout the day/night cycle by stomatal closure and a highly impervious cuticle. The hypothesis is that endogenously produced CO₂ is retained and recycled through dark CO₂ fixation, organic acid transformations, photosynthesis, and respiration. Immediately following precipitation, nighttime stomatal opening is initiated, permitting increased atmospheric CO₂ assimilation and organic acid synthesis.     

Photosynthesis in Isolated Chloroplasts of the Crassulacean Acid Metabolism Plant Sedum praealtum

Intact chloroplasts were isolated from protoplasts of the Crassulacean acid metabolism plant Sedum praealtum D.C. Typical rates of CO₂ fixation or CO₂-dependent O₂ evolution ranged from 20 to 30 micromoles per milligram chlorophyll per hour and could be stimulated 30 to 50% by several Calvin cycle intermediates. The pH optimum for CO₂ fixation was 7.0 to 7.6 with considerable activity as low as pH 6.4. Low concentrations of orthophosphate (Pi) (optimum 0.4 millimolar) stimulated photosynthesis while high concentrations (5 millimolar) caused some inhibition. Both CO₂ fixation and CO₂-dependent O₂ evolution exhibited a relatively long lag phase (4 to 6 minutes) which remained constant between 0.4 to 5 millimolar Pi. The lag phase could be decreased by addition of dihydroxyacetone-phosphate or ribose 5-phosphate. Further results are presented which suggest these chloroplasts have a functional phosphate translocator.     

Histochemical Compartmentation of Photosynthesis in the Crassulacean Acid Metabolism Plant Crassula falcata

The succulent leaf of the obligate Crassulacean acid metabolism plant Crassula falcata comprises two distinct types of parenchyma. The peripheral tissue is dark green, whereas the central tissue is relatively colorless. We have investigated whether the conventional interpretation of Crassulacean acid metabolism—simply, temporal separation of light and dark CO₂ fixation within individual cells—is sufficient. Ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) and chlorophyll, indicating the photosynthetic-carbon-reduction pathway, were concentrated in peripheral tissue. Specific activities of P-enolpyruvate carboxylase (4.1.1.31) and of NAD⁺-malic enzyme (1.1.1.39), indicating capacity for dark CO₂ fixation and release, respectively, were high in both types of parenchyma. Measured directly as malic acid decline at the beginning of the photoperiod, CO₂ “storage” occurred in both tissues. These data indicate that there is a spatial component to Crassulacean acid metabolism in C. falcata.     

The Response of Leaf Water Potential and Crassulacean Acid Metabolism to Prolonged Drought in Sedum rubrotinctum

Plants of Sedum rubrotinctum R. T. Clausen were studied in a green-house over a 2-year period without watering. Only the apical leaves survived and were turgid at the end of the experiment. The midday leaf water potential of these apical leaves was −1.20 megapascals, while the leaf water potential of comparable leaves on well-watered control plants was −0.20 megapascals. The unwatered plants appear to have maintained turgor by means of an osmotic adjustment. After 2 years without water the plants no longer exhibited a nocturnal accumulation of titratable acidity. However, the daytime levels of titratable acidity of the unwatered plants were more than 2-fold greater than the levels in well-watered control plants. Well-watered plants of S. rubrotinctum exhibited seasonal shifts in biomass stble carbon isotope ratios, indicating a greater proportion of day versus night CO₂ uptake in the winter than in the summer. The imposition of water stress prevented the expression of this seasonal rhythm and restricted the plants to dark CO₂ uptake.     

Seasonal Patterns of Acid Metabolism and Gas Exchange in Opuntia basilaris

Acid metabolism and gas exchange studies were conducted in situ on the cactus Opuntia basilaris Engelm. and Bigel. A pattern of significant seasonal variation was evident. The pattern was controlled by rainfall, which significantly influenced plant water potentials, total gas transfer resistances, and nocturnal organic acid synthesis. In winter and early spring, when plant water stress was mild, stomatal and mesophyll resistances remained low, permitting enhanced nocturnal assimilation of ¹⁴CO₂. The day/night accumulation of acidity was large during these seasons. In summer and fall, plant water stress was moderate, although soil water stress was severe. The nocturnal assimilation of ¹⁴CO₂ was very low during these seasons, even in stems with open stomata, indicating large mesophyll resistances restricting exogenous gas incorporation. The day/night accumulation of acidity was reduced, and a low level of acid metabolism persisted throughout this period. The rapid response to a midsummer rainfall emphasizes the importance of plant water potential as a parameter controlling over-all metabolic activity. The seasonal variations of acid metabolism and gas exchange significantly influenced the efficiency of water use and carbon dioxide assimilation. Periods of maximal efficiency followed rainfall throughout the course of the year.     

Malate Decarboxylation by Mitochondria of the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum

Mitochondria isolated from leaves of Mesembryanthemum crystallinumoxidized malate by both NAD malic enzyme and NAD malate dehydrogenase.Rates of malate oxidation were higher in mitochondria from plantsgrown at 400 mil NaCl in the rooting medium and performing Crassulaceanacid metabolism (CAM) than in mitochondria from plants grownat 20 mM NaCl and exhibiting C₃-photosynthetic CO₂ fixation.The mitochondria isolated from plants both in the CAM and C₃modes were tightly coupled and gave high respiratory control.At optimum pH for malate oxidation (pH 7.0), pyruvate was themajor product in mitochondria from CAM-M. crystallinum, whereasmitochondria from C₃-M. crystallinum produced predominantlyoxaloacetate. Both the extracted NAD malic enzyme in the presenceof CoA and the oxidation of malate to pyruvate by the mitochondriafrom plants in the CAM mode had a pH optimum around 7.0 withactivity declining markedly above this pH. The activity of NAD-malicenzyme, expressed on a cytochrome c oxidase activity basis,was much higher in mitochondria from the CAM mode than the C₃mode. The results indicate that mitochondria of this speciesare adapted to decarboxylate malate at high rates during CAM. ¹Current address: Lehrstuhl für Botanik II, UniversitätWurzburg, Mittlerer Dallenbergweg 64, 8700 Würzburg, WestGermany. ²Current address: KD 120, Chemical Research Division, OntarioHydro, 800 Kipling Avenue, Toronto, Ontario M8Z5S4, Canada. ³Current address: Department of Botany, Washington State University,Pullman, Washington 99164-4230, U.S.A. (Received March 13, 1986; Accepted September 18, 1986)     

Patterns of Carbon Partitioning in Leaves of Crassulacean Acid Metabolism Species during Deacidification

Carbohydrates stored during deacidification in the light were examined in 11 Crassulacean acid metabolism (CAM) species from widely separated taxa grown under uniform conditions. The hypothesis that NAD(P) malic enzyme CAM species store chloroplastic starch and glucans, and phosphoenolpyruvate carboxykinase species store extrachloroplastic sugars or polymers was disproved. Of the six malic enzyme species examined, Kalanchoe tubiflora, Kalanchoe pinnata, Kalanchoe daigremontiana, and Vanilla planifolia stored mainly starch. Sansevieria hahnii stored sucrose and Agave guadalajarana did not store starch, glucose, fructose, or sucrose. Of the five phosphoenolpyruvate carboxykinase species investigated, Ananus comosus stored extrachloroplastic carbohydrate, but Stapelia gigantea, Hoya carnosa, and Portea petropolitana stored starch, whereas Aloe vera stored both starch and glucose. Within families, the major decarboxylase was common for all species examined, whereas storage carbohydrate could differ both between and within genera. In the Bromeliaceae, A. comosus stored mainly fructose, but P. petropolitana stored starch. In the genus Aloe, A. vera stored starch and glucose, but A. arborescens is known to store a galactomannan polymer. We postulate that the observed variation in carbohydrate partitioning between CAM species is the result of two principal components: (a) constraints imposed by the CAM syndrome itself, and (b) diversity in biochemistry resulting from different evolutionary histories.     

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

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

Effects of seasonal changes in the Midwest on Crassulacean Acid Metabolism (CAM) in Opuntia humifusa Raf

Summary Seasonal changes in the Crassulacean Acid Metabolism (CAM) activity and growth characteristics of Opuntia humifusa Raf. were examined under midwest climatic conditions. Twenty-four hour studies were done at monthly intervals for two years, with diurnal changes in transpiration, gas exchange, and titratable acidity monitored under natural conditions. CAM activity was observed only from April to September, but occurred regardless of changes in temperature or precipitation. The maximum rate of dark CO₂ uptake occurred in May, while greatest acid fluctuations coincided with flowering, new growth, and high tissue water content in June. In spite of conditions favorable for CAM, acidification and rate of dark CO₂ assimilation decreased progressively through September as stem water content dropped and shoot production ceased. No CAM was evident during sub-zero winter months and tissue water content decreased to only 65% (<-20 bars). Winter survival of plants in test plots was found to be affected by the amount of water received the preceding growing season; the driest group showing the lowest mortality rate.Although spring and autumn were considered periods likely to exhibit CAM, it was not observed during those months just prior to or immediately following winter (Oct. and March). Acid fluctuations were minimal with CO₂ being taken up during the day and released at night, indicating some degree of flexibility in the CAM activity of Opuntia humifusa.This study supported by Sigma XI Research Society of North America, and by National Science Foundation Grants PCM-77-25100 and PCM-79-05937     

Gas Exchange Characteristics of the Submerged Aquatic Crassulacean Acid Metabolism Plant, Isoetes howellii

The submerged aquatic plant Isoetes howellii Engelmann possesses Crassulacean acid metabolism (CAM) comparable to that known from terrestrial CAM plants. Infrared gas analysis of submerged leaves showed Isoetes was capable of net CO₂ uptake in both light and dark. CO₂ uptake rates were a function of CO₂ levels in the medium. At 2,500 microliters CO₂ per liter (gas phase, equivalent to 1.79 milligrams per liter aqueous phase), Isoetes leaves showed continuous uptake in both the light and dark. At this CO₂ level, photosynthetic rates were light saturated at about 10% full sunlight and were about 3-fold greater than dark CO₂ uptake rates. In the dark, CO₂ uptake rates were also a function of length of time in the night period. Measurements of dark CO₂ uptake showed that, at both 2,500 and 500 microliters CO₂ per liter, rates declined during the night period. At the higher CO₂ level, dark CO₂ uptake rates at 0600 h were 75% less than at 1800 h. At 500 microliters CO₂ per liter, net CO₂ uptake in the dark at 1800 h was replaced by net CO₂ evolution in the dark at 0600 h. At both CO₂ levels, the overnight decline in net CO₂ uptake was marked by periodic bursts of accelerated CO₂ uptake. CO₂ uptake in the light was similar at 1% and 21% O₂, and this held for leaves intact as well as leaves split longitudinally. Estimating the contribution of light versus dark CO₂ uptake to the total carbon gain is complicated by the diurnal flux in CO₂ availability under field conditions.     

Diurnal Ion Fluctuations in the Mesophyll Tissue of the Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum

Both laboratory- and field-grown Mesembryanthemum crystallinum plants exhibited large scale diurnal ion fluctuations. In mesophyll tissue, potassium and sodium levels varied in conjunction with acid levels while chloride levels varied in opposition. Thus, dark CO₂ fixation in this Crassulacean acid metabolism species seems analogous to the common plant process of malate synthesis to balance cation surplus. Sodium levels in the epidermis appeared to fluctuate in opposition to those in the mesophyll. It is proposed that inorganic cations cycle between mesophyll and epidermal tissue to balance malate accumulation and to produce stomatal opening in the dark.     

Characterization of Early Morning Crassulacean Acid Metabolism in Opuntia erinacea var Columbiana (Griffiths) L. Benson

The nature and sequence of metabolic events during phase II (early morning) Crassulacean acid metabolism in Opuntia erinacea var columbiana (Griffiths) L. Benson were characterized. Gas exchange measurements under 2 and 21% O₂ revealed increased O₂ inhibition of CO₂ fixation with progression of phase II. Malate and titratable acidity patterns indicated continued synthesis of C₄ acids for at least 30 minutes into the light period. Potential activities of phosphoenolpyruvate carboxylase (PEPC) and NADP-malic enzyme exhibited little change during phase II, while light activation of NADP-malate dehydrogenase, pyruvate, orthophosphate dikinase, and ribulose-1,5-bisphosphate carboxylase was apparent. Short-term ¹⁴CO₂ fixation experiments showed that the per cent of ¹⁴C incorporated into C₄ acids decreased while incorporation into other metabolites increased with time. PEPC exhibited increased sensitivity to 2 millimolar malate, and the K_i(malate) for PEPC decreased markedly with time. Sensitivity of PEPC to malate inhibition was considerably greater at pH 7.5 than at 8.0. The results indicate that decarboxylation and synthesis of malate occur simultaneously during the early morning period, and that phase II acid metabolism is not limited by CO₂ diffusion through stomata. With progression of phase II, CO₂ fixation by PEPC decreases while fixation by ribulose-1,5-bisphosphate carboxylase increases.     

Effect of Severe Water Stress on Aspects of Crassulacean Acid Metabolism in Xerosicyos

Xerosicyos danguyi H.Humb. (Cucurbitaceae) is a Crassulacean acid metabolism (CAM) species native to Madagascar. Previously, it was shown that when grown under good water conditions, it is a typical CAM plant, but when water stressed, it shifts to a dampened form of CAM, termed CAM-idling, in which stomata are closed day and night but with a continued, low diurnal organic acid fluctuation. We have now studied the kinetics of some metabolic features of the shift from CAM to CAM-idling under severe water stress and the recovery upon rewatering. When water is withheld, there is a steady decrease in relative water content (RWC), reaching about 50%, at which point the water potential decreases precipitously from about -2 or -3 bars to -12 bars. Abscisic acid (ABA) increases sharply at about 75% RWC. Stomata close, which limits CO2 uptake, and there is a dampened diurnal organic acid fluctuation typical of CAM-idling. Throughout an extended stress period to 50% RWC, there is no change in chlorophyll, protein, and ribulose bisphosphate carboxylase activity compared with the well-watered plants. Despite the fact that the tissue was already in CAM, the stress is accompanied by an increase in phosphoenolpyruvate carboxylase (PEPc) mRNA, extractable PEPc activity, and PEPc protein (such that the specific activity remained approximately constant) and a decrease in the apparent Km(PEP). It is not known if the changes in Km(PEP) in response to drought are related to or are separate from the increases in PEPc protein and mRNA. The changes in Km(PEP) could be in response to the decreased endogenous levels of organic acids, but evidently are not an assay artifact. The increases in PEPc protein and mRNA appear to be related to the water-stress treatment and may result from the increased concentration of ABA or the decreased levels of endogenous organic acids. When rewatered, the metabolism quickly returns to the well-watered control typical of CAM.     

Environmental Influences on Open Stomates of a Crassulacean Acid Metabolism Plant, Agave deserti

The major short term stomatal response of Agave deserti was to temperature; increases in leaf temperature led to decreases in water vapor conductance for stomatal opening during the daytime (C₃ mode) as well as at night (Crassulacean acid metabolism or CAM mode). Hourly changes in the water vapor concentration drop from leaf to air had no significant stomatal effect in either mode. Stomatal responses to external CO₂ levels up to 800 microliters per liter were not significant after 15 minutes and only moderate after a few hours, suggesting that CO₂ effects on open stomates of this succulent were indirect in both CAM and C₃ modes.     

Shrinkage of Attached Roots of Opuntia ficus-indica in Response to Lowered Water Potentials--Predicted Consequences for Water Uptake or Loss to Soil

Attached 2-month-old roots of the succulent plant, Opuntia ficus-indica,shrank 0.4% radially during periods of maximal transpirationunder wet conditions. In contrast, reversible decreases in diameterof nearly 20% occurred for these roots as their ambient waterpotential () in the vapour phase decreased from –0.01to –10 MPa over 8 d, the changes being slightly more rapidat 40 °C than at 10 °C. Such substantial diameter changesbecame progressively less with root age, from a 43% decreasein diameter at 3 weeks to a 6% decrease at 12 months Root shrinkagewas slight when was decreased from –0.01 to –0.3MPa, the latter being similar to the root water potential.As was further decreased from –0.3 to –10 MPa,water movement out of cortical cells caused considerable rootshrinkage. The root hydraulic conductivity (L_p) decreased only30 to 60% for a change in from –0.01 to –10 MPacompared with a decrease of over 10⁶-fold for the soil hydraulicconductivity over this range. The overall conductivity of thesoil, the root-soil air gap, and the root was predicted to bedominated by L_p for _soil above –0.3 MPa. As simulated_soil decreased below –0.3 MPa, the root-soil air gap initiallybecame the primary limiter of water loss from the roots. Below–5 MPa for 1-month-old roots and below –2 MPa for12-month-old roots, the soil became the main limiter of waterloss. Thus, water uptake from wet soils apparently was mainlycontrolled by root properties Water loss to drying soils wascontrolled by the development of a root-soil air gap aroundshrinking roots during the initial phase of soil drying andby the reduction of the soil hydraulic conductivity at evenlower _soil. Root diameter, root hydraulic conductivity, root-soil air gap, soil hydraulic conductivity     

Light and Temperature Regulation of Early Morning Crassulacean Acid Metabolism in Opuntia erinacea var Columbiana (Griffiths) L. Benson

During the early morning period, light and temperature exert distinctively different influences on the gas exchange patterns of the Crassulacean acid metabolism plant Opuntia erinacea through their effects on acid metabolism. An initial decrease in CO₂ uptake was triggered by illumination and was apparently due to a decreased CO₂ diffusion gradient through light-mediated decarboxylation of malate. In contrast, the morning burst of CO₂ uptake occurred at high temperature presumably in response to increases in both stomatal conductance and the CO₂ diffusion gradient, resulting from the temperature-regulated fixation of endogenous CO₂, primarily into malate. Subsequent stomatal closure, apparently due to elevated levels of internal CO₂ through rapid decarboxylation of malate at high temperature, was primarily responsible for the final termination of early morning Crassulacean acid metabolism.     

Carbon Flow and Metabolic Specialization in the Tissue Layers of the Crassulacean Acid Metabolism Plant, Peperomia camptotricha

Leaves of Peperomia camptotricha contain three distinct upper tissue layers and a one-cell thick lower epidermis. Light and dark CO₂ fixation rates and the activity of ribulose bisphosphate carboxylase/oxygenase and several C₄ enzymes were determined in the three distinct tissue layers. The majority of the C₄ enzyme activity and dark CO₂ fixation was associated with the spongy mesophyll, including the lower epidermis; and the least activity was found in the median palisade mesophyll. In contrast, the majority of the C₃ activity, that is ribulose bisphosphate carboxylase/oxygenase and light CO₂ fixation, was located in the palisade mesophyll. In addition, the diurnal flux in titratable acidity was greatest in the spongy mesophyll and lowest in the palisade mesophyll. The spatial separation of the C₃ and C₄ phases of carbon fixation in P. camptotricha suggests that this Crassulacean acid metabolism plant may have low photorespiratory rates when it exhibits daytime gas exchange (that is, when it is well watered). The results also indicate that this plant may be on an evolutionary path between a true Crassulacean acid metabolism plant and a true C₄ plant.