Differential expression of photosynthesis genes in leaf tissue layers of Peperomia as revealed by tissue printing

Peperomia has species that may be C₃, show Crassulacean acid metabolism (CAM), or CAM-cycling. Species that show CAM progress from C₃ to CAM through CAM-cycling during leaf development. In CAM and CAM-cycling species, CAM metabolism is predominately in the upper multiple epidermis and lower spongy mesophyll, whereas C₃ metabolism is localized mostly in the palisade mesophyll. Using specific protein and cDNA probes prepared from P-enolpyruvate carboxylase (PEPc) and ribulose-1,5-bisphosphate carboxylase (Rubisco), we have now studied the differential distribution of photosynthetic metabolism in Peperomia leaves using the technique of tissue printing. The tissue printing studies detected Rubisco protein in leaves of C₃ P. orba, but not PEPc. Young C₃ leaves of P. scandens and P. camptotricha showed Rubisco protein, but not PEPc; however, the mature leaves of these two species that have CAM showed PEPc protein and RNAs in both the multiple epidermis and spongy mesophyll. In contrast, Rubisco protein and RNAs were present throughout the leaf. The tissue printing data are consistent with our previously published data showing the differential distribution of photosynthetic metabolism in leaves of Peperomia. Although the tissue printing technique is qualitative, coupled with quantitative data it has proven useful for the study of function related to structure.     

Photosynthetic Characteristics of the Palisade Mesophyll and Spongy Mesophyll in the CAM/C4 Intermediate Plant,Peperomia camptotricha*

Several photosynthetic parameters were examined in the different tissue layers of leaves from the recently characterized crassulacean acid metabolism/C₄ intermediate plant, Peperomia camptotricha (Nishio and Ting, 1987). Light appears to control the development of certain photosynthetic characteristics within the tissue layers, while factors other than light seem to dictate others. Analysis of the chlorophyll content (including P700) and chlorophyll-proteins indicated that more light harvesting chlorophylls were associated with reaction centers in the tissues that were shaded by overlying tissue. Electron transport activity and chlorophyll-protein analysis indicated that the ratio of photosystem I to photosystem II in the spongy mesophyll (the abaxial tissue type and hence most shaded) was elevated relative to the overlying chlorophyll-rich median palisade mesophyll. The elevated photosystem I relative to photosystem II in the spongy mesophyll of Peperomia camptotricha may be related to C₄ metabolism and an increased requirement for ATP.     

Relationship between Respiration and CAM-Cycling in Peperomia camptotricha

Mature leaves of well-watered Peperomia camptotricha show Crassulacean acid metabolism (CAM). Young leaves show CAM-cycling in which CO₂ uptake occurs during the day concomitant with a marked diurnal fluctuation of organic acids as in CAM. Evidence is presented suggesting that respiration is the source of CO₂ for nocturnal acid synthesis in leaves exhibiting CAM-cycling. Respiratory quotients for these leaves were consistently much less than unity despite the fact that the leaves metabolize starch. The conservation of CO₂ by refixation into acids at night represents about 17% of the total photosynthetically fixed CO₂ and about 50% of the total respiratory CO₂.     

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.     

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.     

CRASSULACEAN ACID METABOLISM IN THE GESNERIACEAE

The occurrence of the Crassulacean acid metabolism (CAM) was studied in four epiphytic species of the Gesneriaceae: two neotropical species, Codonanthe crassifolia and Columnea linearis, and two paleotropical species, Aeschynanthus pulcher and Saintpaulia ionantha. Gas exchange parameters, enzymology, and leaf anatomy, including mesophyll succulence and relative percent of the mesophyll volume occupied by airspace, were studied for each species. Codonanthe crassifolia was the only species to show nocturnal CO₂ uptake and a diurnal organic acid fluctuation. According to these results, Codonanthe crassifolia shows CAM-cycling under well-watered conditions and when subjected to drought, it switches to CAM-idling. Other characteristics, such as leaf anatomy, mesophyll succulence, and PEP carboxylase and NADP malic enzyme activity, indicate attributes of the CAM pathway. All other species tested showed C₃ photosynthesis. The most C₃-like species is Columnea linearis, according to the criteria tested in this investigation. The other two species show mesophyll succulence and relative percent of the leaf volume occupied by airspace within the CAM range, but no other characters of the CAM pathway. The leaf structure of certain genera of the Gesneriaceae and of the genus Peperomia in the Piperaceae are similar, both having an upper succulent, multiple epidermis, a medium palisade of one or a few cell layers, and a lower, succulent spongy parenchyma not too unlike CAM photosynthetic tissue. We report ecophysiological similarities between these two distantly related families. Thus, the occurrence of CAM-cycling may be more common among epiphytic species than is currently known.     

Ecophysiology of the C3-CAM intermediate Clusia minor L. in Trinidad: seasonal and short-term photosynthetic characteristics of sun and shade leaves

The seasonal changes in crassulacean acid metabolism (CAM) activity in response to daily integrated photon flux density (PFD) and precipitation were compared in sun and shade leaves of the C3-CAM intermediate tree Clusia minor L. Measurements of CAM activity (H⁺) showed that maximum leaf acidity consistently occurred 4 h after dawn, suggesting that new sampling procedures need to be adopted in order to quantify CAM in Clusia species. Whilst exposed leaves responded to intermittent dry conditions, shaded leaves showed a clear induction of CAM activity as conditions became drier. The magnitude of CAM activity correlated well with daily integrated PFD, such that the extent of decarboxylation of organic acids was consistently associated with increased acidification during the subsequent dark period. Over two sampling days, both sun and shade leaves exhibited the four phases of CAM, although PEPc remained active throughout phase II with the result that 50% of the maximum leaf acidity in shade leaves was accumulated during this time. During phase III, internal CO2 supply was augmented by substantial citrate decarboxylation, in addition to malic acid. Chlorophyll fluorescence characteristics were dominated by high rates of PSII electron transport, together with an extremely high potential for thermal dissipation, such that excess light was maintained within safe limits at times of maximum PFD. Photochemical stability was maintained by matching supply and demand for internal CO2: in the morning, C3 and C4 carboxylation processes were regulated by extended PEPc activity, so that decarboxylation was delayed until temperature and light stress were highest at midday.     

Variable Photosynthetic Metabolism in Leaves and Stems of Cissus quadrangularis L

By measuring titratable acidity, gas exchange parameters, mesophyll succulence, and ¹³C/¹²C ratios, we have shown that Cissus quadrangularis L. has C₃-like leaves and stems with Crassulacean acid metabolism (CAM). In addition, the nonsucculent leaves show the diurnal fluctuations in organic acids termed recycling despite the fact that all CO₂ uptake and stomatal opening occurs during the day. Young succulent stems have more C₃ photosynthesis than older stems, but both have characteristics of CAM. The genus Cissus will be a fruitful group to study the physiology, ecology, and evolution of C₃ and CAM since species occur that exhibit characteristics of both photosynthetic pathways.     

Crassulacean Acid Metabolism and Crassulacean Acid Metabolism Modifications in Peperomia camptotricha

Peperomia camptotricha, a tropical epiphyte from Mexico, shows variable forms of Crassulacean acid metabolism (CAM). Young leaves exhibit CAM-cycling, while mature leaves show an intermediate type of metabolism, between CAM and CAM-cycling, having approximately the same amount of nighttime gas exchange as daytime. Metabolism of young leaves appears independent of daylength, but mature leaves have a tendency toward more CAM-like metabolism under short days (8 hours). Large differences in the physical appearance of plants were found between those grown under short daylengths and those grown under long daylengths (14 hours). Some anatomical differences were also detected in the leaves. Water stress caused a switch to CAM in young and mature leaves, and as water stress increased, they shifted to CAM-idling.     

Succulence and CAM relationships in Aeonium genus

The CAM has been tested in six species of the Aeonium genus by studying the diurnal fluctuation of organic acids, pH and night fixation of CO₂. The existence of a mesophyll structure able to support this metabolism has been shown as well as a congruent periodicity in the pool of cell starch. We have calculated the S, ES and Sm indices in the six species. A series of regression equations of different grades and types were calculated and shown to have correlation coefficients statistically significant. This allows us to confirm the suitability of the Sm index as a rapid test to establish the CAM as postulated by former authors.     

Developmental Control of CAM in Peperomia scandens

Experiments were conducted to examine the development of photosynthetic carbon metabolism in Peperomia scandens, a tropical epiphyte. Leaves were sampled during a 10-day period when they were between 30 to 165 days old. P. scandens exhibits a C₃ to CAM-cycling to CAM shift during maturation with the magnitude of CAM increasing with age. Initially, during both day and night, no significant CO₂ uptake or diurnal acid flux was evident. C₃ gas exchange was detected at 41 days of age with a gradual shift towards CAM gas exchange maximized thereafter. An acidity flux of 130 to 150 microequivalents per gram fresh weight was evident by 41 days. Between 40 and 90 days, the leaves shifted their CO₂ uptake pattern from a daytime to a nighttime peak. After 90 days, the leaves remained in CAM. The δ¹³C values became progressively less negative as the leaves matured. In the 30-day-old leaves, the δ¹³C value was −21.1% while in the 165-day-old leaves the δ¹³C value was −18.3%. The time-dependent shift from C₃ to CAM-cycling to CAM in P. scandens does not appear to result from changes in water, light, or temperature regimes since these variables were constant for all leaves sampled.     

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.     

Inducibility of crassulacean acid metabolism (CAM) in Clusia species; physiological/biochemical characterisation and intercellular localization of carboxylation and decarboxylation processes in three species which exhibit different degrees of CAM

The biochemical basis for photosynthetic plasticity in tropical trees of the genus Clusia was investigated in three species that were from contrasting habitats and showed marked differences in their capacity for crassulacean acid metabolism (CAM). Physiological, anatomical and biochemical measurements were used to relate changes in the activities/amounts of key enzymes of C₃ and C₄ carboxylation to physiological performance under severe drought stress. On the basis of gas-exchange measurements and day/night patterns of organic acid turnover, the species were categorised as weak CAM-inducible (C.aripoensis Britt.), C₃-CAM intermediate (C. minor L.) and constitutive CAM (C.␣rosea Jacq. 9.). The categories reflect genotypic differences in physiological response to drought stress in terms of net carbon gain; in C. aripoensis net carbon gain was reduced by over 80% in drought-stressed plants whilst carbon gain was relatively unaffected after 10 d without water in C. rosea. In turn, genotypic differences in the capacity for CAM appeared to be directly related to the capacities/amounts of phosphoenolpyruvate carboxylase (PEPCase) and phosphoenolpyruvate carboxykinase (PEPCK) which increased in response to drought in both young and mature leaves. Whilst measured activities of PEPCase and PEPCK in well-watered plants of the C₃-CAM intermediate C. minor were 5–10 times in excess of that required to support the magnitude of organic acid turnover induced by drought, close correlations were observed between malate accumulation/PEPCase capacity and citrate decarboxylation/PEPCK capacity in all the species. Drought stress did not affect the amount of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) protein in any of the species but Rubisco activity was reduced by 35% in the weak CAM-inducible C. aripoensis. Similar amounts of glycine decarboxylase (GDC) protein were present in all three species regardless of the magnitude of CAM expression. Thus, the constitutive CAM species C. rosea did not appear to show reduced activity of this key enzyme of the photorespiratory pathway, which, in turn, may be related to the low internal conductance to CO₂ in this succulent species. Immuno-histochemical techniques showed that PEPCase, PEPCK and Rubisco were present in cells of the palisade and spongy parenchyma in leaves of species performing CAM. However, in leaves from well-watered plants of C. aripoensis which only performed C₃ photosynthesis, PEPCK was localized around latex-producing ducts. Differences in leaf anatomy between the species suggest that the association between mesophyll succulence and the capacity for CAM in these hemi-epiphytic stranglers has been selected for in arid environments. Received: 4 July 1997 / Accepted: 27 November 1997     

Higher plant phosphoenolpyruvate carboxylase kinase is regulated at the level of translatable mRNA in response to light or a circadian rhythm

Phosphoenolpyruvate carboxylase is regulated by reversible phosphorylation in response to light in C₃ and C₄ plants and to a circadian oscillator in CAM plants. Increases in phosphoenolpyruvate carboxylase kinase activity require protein synthesis. This requirement has been analysed by quantifying translatable mRNA for this protein kinase using in vitro translation of isolated RNA followed by direct assay of kinase activity. In leaves of the CAM plant Bryophyllum (Kalanchoë) fedtschenkoi, in normal diurnal conditions, kinase mRNA was 20-fold more abundant at night than in the day. In constant environmental conditions (continuous darkness, CO₂-free air, 15°C) kinase mRNA exhibited circadian oscillations. The circadian disappearance of kinase mRNA and kinase activity was delayed by lowering the temperature to 4°C and accelerated by raising the temperature to 30°C. The appearance of kinase mRNA and activity was blocked by cordycepin and puromycin. In maize and barley, kinase mRNA increased in response to light. For all three plants, the phosphoenolpyruvate carboxylase kinase activity generated during in vitro translation was Ca²⁺-independent. These results demonstrate that phosphoenolpyruvate carboxylase kinase activity is regulated at the level of translatable mRNA in C₃, C₄ and CAM plants.     

Structural Adaptation of the Leaf Mesophyll to Shading

Structural characteristics of the mesophyll were studied in five boreal grass species experiencing a wide range of light and water supply conditions. Quantitative indices of the palisade and spongy mesophyll tissues (cell and chloroplast sizes, the number of chloroplasts per cell, the total cell and chloroplast surface area per unit leaf surface area) were determined in leaves of each of the species. The cell surface area and the cell volume in spongy mesophyll were determined with a novel method based on stereological analysis of cell projections. An important role of spongy parenchyma in the photosynthetic apparatus was demonstrated. In leaves of the species studied, the spongy parenchyma constituted about 50% of the total volume and 40% of the total surface area of mesophyll cells. The proportion of the palisade to spongy mesophyll tissues varied with plant species and growth conditions. In a xerophyte Genista tinctoria, the total cell volume, cell abundance, and the total surface area of cells and chloroplasts were 30–40% larger in the palisade than in the spongy mesophyll. In contrast, in a shade-loving species Veronica chamaedris, the spongy mesophyll was 1.5–2 times more developed than the palisade mesophyll. In mesophyte species grown under high light conditions, the cell abundance and the total cell surface area were 10–20% greater in the palisade mesophyll than in the spongy parenchyma. In shaded habitats, these indices were similar in the palisade and spongy mesophyll or were 10–20% lower in the palisade mesophyll. In mesophytes, CO₂ conductance of the spongy mesophyll accounted for about 50% of the total mesophyll conductance, as calculated from the structural characteristics, with the mesophyll CO₂ conductance increasing with leaf shading.     

Kinetic Properties of Phosphoenolpyruvate Carboxylase in Peperomia camptotricha

Kinetic characteristics of phosphoenolpyruvate carboxylase (PEPC) from the epiphytic C₃ or C₄: CAM intermediate plant, Peperomia camptotricha, were investigated. Few day versus night differences in V_max,K_m(PEP)), or malate inhibition were observed, even in extracts from water-stressed plants which characteristically perform CAM, regardless of efforts to stabilize day/night forms. The PEPC extracted from plants during the light period remained stable, without much of an increase or decrease in activity for at least 22 hours at 0 to 4°C. Extracts from mature, fully developed leaves had slightly greater PEPC activity than from very young, developing leaves. Generally, however, the kinetic properties of PEPC extracted from mature leaves of plants grown under short day (SD), long day (LD), or 1-week water-stress conditions, as well as from young, developing leaves, were similar. The PEPC inhibitor, l-malate, decreased the V_max and increased the K_m(PEP) for all treatments. Under specific conditions, malate did not inhibit PEPC rates in the dark extracts as much as the light. The PEPC activator, glucose-6-phosphate (G-6-P), lowered the K_m(PEP) for all treatments. At saturating PEP concentrations, PEPC activity was independent of pH in the range of 7.5 to 9.0. At subsaturating PEP concentrations, the pH optimum was 7.8. The rates of PEPC activity were lower in the light period extracts than the dark, at pH 7.1, but day/night PEPC was equally active at pH 7.8. At pH 7.5 and a subsaturating PEP concentration, G-6-P significantly activated PEPC. At pH 8, however, only slight activation by G-6-P was observed. The lower pH of 7.5 combined with l-malate addition, greatly inhibited PEPC, particularly in extracts from young, developing leaves which were completely inhibited at an l-malate concentration of 1 millimolar. However, malate did not further inhibit PEPC activity in mature leaves when assayed at pH 7.1. The fairly constant day/night kinetic and regulatory properties of PEPC from P. camptotricha are unlike those of PEPC from CAM or C₄ species studied, and are consistent with the photosynthetic metabolism of this plant.     

Effects of Various Levels of CO(2) on the Induction of Crassulacean Acid Metabolism in Portulacaria afra (L.) Jacq

In response to water stress, Portulacaria afra (L.) Jacq. (Portulacaceae) shifts its photosynthetic carbon metabolism from the Calvin-Benson cycle for CO₂ fixation (C₃) photosynthesis or Crassulacean acid metabolism (CAM)-cycling, during which organic acids fluctuate with a C₃-type of gas exchange, to CAM. During the CAM induction, various attributes of CAM appear, such as stomatal closure during the day, increase in diurnal fluctuation of organic acids, and an increase in phosphoenolpyruvate carboxylase activity. It was hypothesized that stomatal closure due to water stress may induce changes in internal CO₂ concentration and that these changes in CO₂ could be a factor in CAM induction. Experiments were conducted to test this hypothesis. Well-watered plants and plants from which water was withheld starting at the beginning of the experiment were subjected to low (40 ppm), normal (ca. 330 ppm), and high (950 ppm) CO₂ during the day with normal concentrations of CO₂ during the night for 16 days. In water-stressed and in well-watered plants, CAM induction as ascertained by fluctuation of total titratable acidity, fluctuation of malic acid, stomatal conductance, CO₂ uptake, and phosphoenolpyruvate carboxylase activity, remained unaffected by low, normal, or high CO₂ treatments. In well-watered plants, however, both low and high ambient concentrations of CO₂ tended to reduce organic acid concentrations, low concentrations of CO₂ reducing the organic acids more than high CO₂. It was concluded that exposing the plants to the CO₂ concentrations mentioned had no effect on inducing or reducing the induction of CAM and that the effect of water stress on CAM induction is probably mediated by its effects on biochemical components of leaf metabolism.     

Evidence that the induction of crassulacean acid metabolism by water stress in Mesembryanthemum crystallinum (L.) involves root signalling

The aim of this study was to investigate whether the root system of Mesembryanthemum crystallinum (L.) plays a role in triggering the induction of crassulacean acid metabolism (CAM) during water stress. Depriving well-irrigated plants of water, by allowing the soil surrounding the roots to dry, caused increased daily losses in leaf relative water content (RVVC) and mesophyll cell turgor pressure. The RWC of the roots also declined. Subsequently plants exhibited physiological characteristics of CAM photosynthesis (i.e. diurnal fluctuations in leaf titratable acidity and nocturnal net CO₂ fixation). When the root system of plants was divided equally between two soil compartments and one half deprived of water, plants exhibited physiological characteristics of CAM without prior changes in leaf RWC content or mesophyll cell turgor pressure. Only the RWC of the water-stressed portion of the roots was reduced. These data suggest that in water-stressed plants daily changes in leaf water relations greater than those observed in well-irrigated plants, are not essential to trigger CAM expression. It is probable that a reduction in soil water availability can be perceived by the roots of M. crystallinum and that this information is conveyed to the leaves triggering the transition from C₃ to CAM photosynthesis.     

Responses of succulents to plant water stress

Experiments were performed to test the hypothesis that succulents “shift” their method of photosynthetic metabolism in response to environmental change. Our data showed that there were at least three different responses of succulents to plant water status. When plant water status of Portulacaria afra (L.) Jacq. was lowered either by withholding water or by irrigating with 2% NaCl, a change from C₃-photosynthesis to Crassulacean acid metabolism (CAM) occurred. Fluctuation of titratable acidity and nocturnal CO₂ uptake was induced in the stressed plants. Stressed Peperomia obtusifolia A. Dietr. plants showed a change from C₃-photosynthesis to internal cycling of CO₂. Acid fluctuation commenced in response to stress but exogenous CO₂ uptake did not occur. Zygocactus truncatus Haworth plants showed a pattern of acid fluctuation and nocturnal CO₂ uptake typical of CAM even when well irrigated. The cacti converted from CAM to an internal CO₂ cycle similar to Peperomia when plants were water-stressed. Reverse phase gas exchange in succulents results in low water loss to carbon gain. Water is conserved and low levels of metabolic activity are maintained during drought periods by complete stomatal closure and continual fluctuation of organic acids.     

Influence of Photoperiod and Leaf Age on Crassulacean Acid Metabolism in Portulacaria afra (L.) Jacq

The possibility that Crassulacean acid metabolism (CAM) is subject to long day photoperiodic control in Portulacaria afra (L.) Jacq., a facultative CAM plant, was studied. Periodic measurements of ¹⁴CO₂ uptake, stomatal resistance, and titratable acidity were made on plants exposed to long and short day photoperiods. Results indicates that waterstressed P. afra had primarily nocturnal CO₂ uptake, daytime stomatal closure, and a large diurnal acid fluctuation in either photoperiod. Mature leaf tissue from nonstressed plants under long days exhibited a moderate diurnal acid fluctuation and midday stomatal closure. Under short days, there was a reduced diurnal acid fluctuation in mature leaf tissue. Young leaf tissue taken from nonstressed plants did not utilize the CAM pathway under either photoperiod as indicated by daytime CO₂ uptake, lack of diurnal acid fluctuation, and incomplete daytime stomatal closure.

The induction of CAM in P. afra appears to be related to the water status of the plant and the age of the leaf tissue. The photosynthetic metabolism of mature leaves may be partly under the control of water stress and of photoperiod, where CAM is favored under long days.