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

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

Comparative ecophysiology of CAM and C3 bromeliads. IV. Plant water relations

Abstract An investigation was carried out into the water relations of CAM and C₃ bromeliads in their natural habitat during the dry season in Trinidad. Measurements were made of xylem tension with the pressure chamber and of cell-sap osmotic pressure and titratable acidity on crushed leaf samples. A steady-state CO₂ and H₂O-vapour porometer was also used so that changes in leaf water relations during individual day-night cycles could be directly related to gas-exchange patterns in situ. Xylem tension changed in parallel with transpiration rate and in general reached its maximum value in CAM bromeliads at night and in C₃ bromeliads during the day. In addition, large nocturnal increases in cell-sap osmotic pressure and titratable acidity (ΔH⁺) typically occurred in the CAM bromeliads. The C₃-CAM intermediate Guzmania monostachia showed slight nocturnal acidification, but had higher values of xylem tension during the day. Very high values of AH+ were observed in the CAM species when the tanks of the epiphytic bromeliads contained water: Aechmea nudicaulis showed a mean maximum ΔH⁺ of 474 mol m^?3, the highest value so far observed for CAM plants. On some nights dew formed on the leaf surfaces of the epiphytes, partially curtailing gas exchange and leading to a marked decrease in xylem tension in both C₃ and CAM species. Between-site comparisons were also made for a wide range of habitats from arid coastal scrub to montane rain forest. Compared with values characteristic of other life-forms, xylem tension and cell-sap osmotic pressure were low for all bromeliads, and did not differ significantly in co-occurring CAM and C₃ bromeliads. Mean maximum xylem tension (10 species in total) ranged from 0.29 M Pa at the montane sites to 0.67 MPa at the most arid site, and mean minimum osmotic pressure (17 species) from 0.51 to 0.97 MPa. At the arid sites the bromeliads were exclusively CAM species, two of which (Aechmea aquilega and Bromelia plumieri) grew terrestrially in the undergrowth of the coastal scrub. Xylem tension in these species was low enough to indicate that they must be functionally independent of the substratum during the dry season. In the wetter part of Trinidad, no between-site differences in leaf water relations were found along an altitudinal gradient in the Northern Mountain Range; seasonal differences in this area were also small. Overall, leaf water relations and gas exchange in the bromeliads were strongly affected both by short-term changes in water availability and by longer-term climatic differences in the various regions of the island.     

Comparative ecophysiology of CAM and C3 bromeliads. I. The ecology of the Bromeliaceae in Trinidad

Abstract This article deals with the physiological ecology of the Bromeliaceae, a large neotropical family containing both terrestrial and epiphytic forms, as well as many species with crassulacean acid metabolism (CAM). The article is in two parts. In the first, we review what is known of the occurrence of CAM and C₃ species in the Bromeliaceae. The photosynthetic pathways are discussed in the context of the major taxonomic divisions within the family and the great diversity of bromeliad life-forms. Of the three subfamilies, the Pitcairnioideae contain both C₃ and CAM species and are essentially all terrestrial. In contrast, the Tillandsioideae are entirely epiphytic or saxicolous, with CAM species being restricted to the genus Tillandsia, And in the Bromelioideae all species show CAM, but terrestrial and epiphytic forms are found in about equal numbers. The evidence suggests that both CAM and the epiphytic habit arose more than once in the family's evolutionary history. In the second part we consider the photosynthetic ecology of the various bromeliad life-forms in more detail using the specific example of Trinidad (West Indies). CAM bromeliads tend to be centred on the drier regions of the island and C₃ forms on the wetter areas. However, at any one site there is a marked vertical stratification of species within the forest profile. Based on the known habitat preferences of the bromeliads, six contrasting sites were selected for field studies in Trinidad. These ranged from arid coastal scrub to montane rain forest, the vegetational and climatic characteristics of which are described here. The constancy of δ¹³C values (carbon-isotope ratios) for individual CAM species in these markedly different habitats emphasized the need for ecophysiological studies to characterize environmental effects on CO₂ assimilation and transpiration. The following papers in this series present the results of a comparative investigation of gas exchange and leaf water relations of CAM and C₃ bromeliads in situ at the various sites.     

The role of CAM in high rainfall cloud forests: an in situ comparison of photosynthetic pathways in Bromeliaceae

Crassulacean acid metabolism (CAM), an advanced photosynthetic pathway conferring water conservation to plants in arid habitats, has enigmatically been reported in some species restricted to extremely wet tropical forests. Of these, epiphytic Bromeliaceae may possess absorbent foliar trichomes that hinder gas‐exchange when wetted, imposing further limitations on carbon dioxide (CO₂) uptake. The hypothesis that the metabolic plasticity inherent to CAM confers an ecological advantage over conventional C₃ plants, when constant rainfall and mist might inhibit gas‐exchange was investigated. Gas‐exchange, fluorometry and organic acid and mineral nutrient contents were compared for the bromeliads Aechmea dactylina (CAM) and Werauhia capitata (C₃) in situ at the Cerro Jefe cloud forest, Panama (annual rainfall > 4 m). Daily carbon gain and photosynthetic nutrient use efficiencies were consistently higher for A. dactylina, due to a greater CO₂ uptake period, recycling of CO₂ from respiration and a dynamic response of CO₂ uptake to wetting of leaf surfaces. During the dry season CAM also had water conserving and photoprotective roles. A paucity of CAM species at Cerro Jefe suggests a recent radiation of this photosynthetic pathway into the wet cloud forest, with CAM extending diversity in form and function for epiphytes.     

Crassulacean acid metabolism in the epiphytic ferns Drymoglossum piloselloides and Pyrrosia longifolia: studies on responses to environmental signals

Abstract The paper reports the results of the comprehensive study of crassulacean acid metabolism in two epiphytic tropical ferns, Drymoglossum piloselloides and Pyrrosia longifolia. The plants were investigated under different light, temperature and water status. It was found that both species are obligate CAM plants. The diurnal acidity rhythm is due to the fluctuation in malic acid concentration, which accounts for the change in titratable acidity. Besides malic acid, shikimate and oxalate are found to be present, but not contributing to the CAM acid rhythm. The diurnal rhythm of malic acid content results in a corresponding rhythm in leaf water relations. Both Φ_Φ and Φ_total, were lowest at the end of the night, i.e. when the level of malic acid was highest. The effects of temperature on CO₂ exchange were inverse to those observed in other CAM plants. In both ferns studied, dark CO₂ fixation increased when the night temperature was increased. Increase in day temperature reduced CO₂ uptake during phase IV and during the following night. The observed responses of the ferns to temperature changes suggest that the in situ environmental conditions are optimal for their CAM performance. In weak light, the plants showed net CO₂ output during the midday deacidification period. Increases in light intensity reduced such CO₂ output. Under drought conditions, the CO₂ exchange in the ferns was reduced to zero within 5–6 d, indicating that the ferns studied are more susceptible to water deficiency than other CAM plants. This could be due to a higher cuticular conductance for water. The results are discussed, in particular, in relation to CAM performance of epiphytes growing in the wet tropics.     

In situ Studies of Crassulacean Acid Metobolism in Drymoglossum piloselloides, an Epiphytic Fern of the Humid Tropics

This paper reports autecological field-studies in Singaporeon Drymoglossum piloselloides (L.) Presl., an epiphytic fernof the humid tropics which is capable of performing Crassulaceanacid metabolism (CAM). As indicated by the gas exchange patternsand by the occurrence of a diurnal malic acid rhythm, the plantalso features CAM in situ at its natural sites. Both in well-wateredand in naturally droughted plants external CO₂ was taken upsolely during the night. Water stress decreased nocturnal CO₂uptake,but left the synthesis and storage of malic acid unaffected.This indicates that CO₂ recycling of respiratory CO₂ by CAMis ecophysiologically important at the high night temperaturestypical of the tropical habitats of the fern. The plants showeda diel fluctuation of cell-sap osmotic pressure which paralleledthat of malic acid, while the fluctuation of the xylem tensionfollowed the curve of transpiration more closely than it followedthat of the malic acid content. CAM in D. piloselloides wasclearly not limited by natural access to mineral ions and nitrogen.It is concluded that the ecophysiological advantage of CAM forD. piloselloides lies in a better water use efficiency as comparedwith C₃ ferns and in the salvaging of carbon by CO₂ recycling. Key words: CAM, epiphytic ferns, gas exchange, water relations     

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

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

Day-night changes in the leaf water relations of epiphytic bromeliads in the rain forests of Trinidad

Summary A study was made of the bulk-leaf water relations of selected species of epiphytic bromeliads growing in their natural habitat in Trinidad (West Indies). Field measurements were made during the rainy season at three forest sites centred on the wetter part of the island. The epiphytic bromeliads were sampled in situ using modified rock-climbing techniques at 4- to 6-h intervals during complete day-nigh cycles. Eleven species were studied that differed in their photosynthetic pathways and habitat preferences.The C₃ species among the epiphytic bromeliads characteristically showed maximum values of xylem tension (measured with the pressure chamber) during the day, whereas the species with crassulacean acid metabolism (CAM) attained maximum values towards the end of the night. In addition, the CAM species showed large nocturnal increases in leaf-cell-sap osmotic pressure and titratable acidity. These nocturnal increases showed mean values of 0.601 MPa and 289 mol H⁺ m^-3, respectively, for four species sampled at an exposed forest clearing (250 m), where CAM species were well represented. At the other two sites, a lowland forest (60 m) and a ridge forest (740 m), CAM bromeliads were found in the forest canopy, but in the lowest strata all the bromeliads were C₃ species. This species distribution was associated with a marked vertical stratification of microlimate, the forest canopy being characterized by much bigger day-night changes in temperature and water-vapour-pressure deficit than the undergrowth. The C₃-CAM intermediateGuzmania monostachia var.monostachia showed significant nocturnal acidification in the forest clearing but not in the understory of the lowland forest.Taken as a whole, the C₃ and CAM bromeliads were very similar in the range of values observed for xylem tension and osmotic pressure, as well as in aspects of their leaf anatomy. However, epidermal trichomes covered a large percentage of the leaf surface area in xeromorphic species (e.g.Tillandsia utriculata), whereas they were poorly developed in shade-tolerant species (e.g.G. lingulata var.lingulata). The absolute values of sylem tension and osmotic pressure were low for all species. Mean minimum xylem tension during the day-night cycles was in the range of 0.18–0.23 MPa and mean maximum in the range 0.41–0.53 MPa; during periods of rain, xylem tension reached a mean minimum of 0.12 MPa. Mean minimum osmotic pressure was in the range 0.449–0.523 MPa. Such between-site and between-species differences as were observed in the water relations of the bromeliads could be related to the microclimatic conditions prevailing in the various epiphytic habitats.Abbreviations CAM crassulacean acid metabolism - PPFD photosynthetic photon flux density     

Canopy CO2 concentrations and Crassulacean acid metabolism in Hoya carnosa in a subtropical rain forest in Taiwan: consideration of CO2 availability and the evolution of CAM in epiphytes

The potential importance of CO₂ derived from host tree respiration at night as a substrate for night time CO₂ uptake during CAM was investigated in the subtropical and tropical epiphytic vine Hoya carnosa in a subtropical rainforest in north-eastern Taiwan. Individuals were examined within the canopies of host trees in open, exposed situations, as well as in dense forests. Although night time CO₂ concentrations were higher near the epiphytic vines at night, relative to those measured during the day, presumably the result of CO₂ added to the canopy air by the host tree, no evidence for substantial use of this CO₂ was found. In particular, stable carbon isotope ratios of H. carnosa were not substantially lower than those of many other CAM plants, as would be expected if host-respired CO₂ were an important source of CO₂ for these CAM epiphytes. Furthermore, laboratory measurements of diel CO₂ exchange revealed a substantial contribution of daytime CO₂ uptake in these vines, which should also result in lower carbon isotope values than those characteristic of a CAM plant lacking daytime CO₂ uptake. Overall, we found that host-respired CO₂ does not contribute substantially to the carbon budget of this epiphytic CAM plant. This finding does not support the hypothesis that CAM may have evolved in tropical epiphytes in response to diel changes in the CO₂ concentrations within the host tree canopy.     

Carbon Assimilation Characteristics of the Aquatic CAM Plant, Isoetes howellii

The relationship between malic acid production and carbon assimilation was examined in the submerged aquatic Crassulacean acid metabolism (CAM) plant, Isoetes howellii Engelmann. Under natural conditions free-CO₂ level in the water was highest at 0600 hours and ¹⁴CO₂ assimilation rates in I. howellii were also highest at this time. After 0900 hours there was a similar pattern in (a) rate of free-CO₂ depletion from the water, (b) reduction of carbon assimilation rates, and (c) rate of deacidification in leaves. Rates of daytime deacidification increased under CO₂-free conditions and as irradiance intensity increased. Nighttime CO₂ uptake was estimated to contribute one-third to one-half of the total daily gross carbon assimilation. CO₂ uptake, however, accounted for only one-third to one-half of the overnight malic acid accumulation. Internal respiratory CO₂ may be a substrate for a large portion of overnight acid accumulation as leaves incubated overnight without CO₂ accumulated substantial levels of malic acid. Loss of CAM occurred in emergent leaf tips even though submerged bases continued CAM. Associated with loss of CAM in aerial leaves was an increase in total chlorophyll, a/b ratio, and carotenoids, and a decrease in leaf succulence. δ¹³C values of I. howellii were not clearly distinguishable from those for associated non-CAM submerged macrophytes.     

Responses of CAM species to increasing atmospheric CO2 concentrations

Crassulacean acid metabolism (CAM) species show an average increase in biomass productivity of 35% in response to a doubled atmospheric CO₂ concentration. Daily net CO₂ uptake is similarly enhanced, reflecting in part an increase in chlorenchyma thickness and accompanied by an even greater increase in water‐use efficiency. The responses of net CO₂ uptake in CAM species to increasing atmospheric CO₂ concentrations are similar to those for C₃ species and much greater than those for C₄ species. Increases in net daily CO₂ uptake by CAM plants under elevated atmospheric CO₂ concentrations reflect increases in both Rubisco‐mediated daytime CO₂ uptake and phosphoenolpyruvate carboxylase (PEPCase)‐mediated night‐time CO₂ uptake, the latter resulting in increased nocturnal malate accumulation. Chlorophyll contents and the activities of Rubisco and PEPCase decrease under elevated atmospheric CO₂, but the activated percentage for Rubisco increases and the K_M(HCO₃ ^? ) for PEPCase decreases, resulting in more efficient photosynthesis. Increases in root:shoot ratios and the formation of additional photosynthetic organs, together with increases in sucrose‐Pi synthase and starch synthase activity in these organs under elevated atmospheric CO₂ concentrations, decrease the potential feedback inhibition of photosynthesis. Longer‐term studies for several CAM species show no downward acclimatization of photosynthesis in response to elevated atmospheric CO₂ concentrations. With increasing temperature and drought duration, the percentage enhancement of daily net CO₂ uptake caused by elevated atmospheric CO₂ concentrations increases. Thus net CO₂ uptake, productivity, and the potential area for cultivation of CAM species will be enhanced by the increasing atmospheric CO₂ concentrations and the increasing temperatures associated with global climate change.     

Alterations in growth and crassulacean acid metabolism (CAM) activity of in vitro cultured cactus

Unlike C-3 plants, cacti possess a crassulacean acid metabolism (CAM) physiology that can alter the pattern of carbon uptake and affect plant growth under artificial environmental conditions, especially in tissue culture. In vitro-derived plantlets of Coryphantha minima grew 7-fold larger than plants cultured under similar ex vitro conditions. Growth regulators incorporated into the culture media during shoot proliferation stage of micropropagation had a strong influence on this increased growth. Other important factors that contributed to increased growth under in vitro conditions were high relative humidity and sugar in the culture medium. An analysis of gas exchange and daily fluctuations of malic acid levels revealed an increase in net photosynthetic rate, in terms of carbon assimilation, by in vitro plants compared with that of ex vitro plants. This stimulated photosynthesis in the presence of an external carbon source was unexpected but apparently true for cacti exhibiting CAM physiology. Unlike CAM plants grown in ex vitro conditions, net CO₂ uptake by in vitro-cultured cacti occurred continuously in the light as well as the dark. Once regenerated, cacti were transferred to ex vitro conditions where the normal CAM pathway resumed with a concomitant reduction in growth and CO₂ uptake. These results showed that growth of cacti can be considerably accelerated by in vitro culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

C3 photosynthesis in the gametophyte of the epiphytic CAM fern Pyrrosia longifolia (Polypodiaceae)

Sporophytes of some epiphytic species in the fern genus Pyrrosia exhibit Crassulacean acid metabolism (CAM), generally considered to be a derived physiological response to xeric habitats. Because these species alternate between independent sporophytic and gametophytic generations yet only the sporophyte has been characterized physiologically, experiments were conducted to determine the photosynthetic pathways present in mature sporophytes, immature sporophytes, and gametophytes of Pyrrosia longifolia. Diurnal CO₂ exchange and malic acid fluctuations demonstrated that although the mature sporophytes exhibited CAM, only C₃ photosynthesis occurred in the gametophytes and young sporophytes. Consideration of the above results and those from previous studies, as well as the life cycle of ferns, indicates that the induction of CAM probably occurs at a certain developmental stage of the sporophyte and/or following exposure to stress. Elucidation of the precise mechanisms underlying this C₃-CAM transition awaits further research.     

Flexibility in CO2 — Fixation Pathway of a Highland Kalanchoë, Kalanchoë petitiana A. Rich.

Photosynthetic flexibility and water use efficiency of Kalanchoë petitiana A. Rich., a facultative CAM plant endemic to the highlands of Ethiopia, were investigated to determine the physiological determinants for the ecological success of the plant. Both field measurements of δ¹³C and greenhouse gas exchange studies showed a shift from C₃ photosynthesis to CAM as leaves aged or at the onset of water stress. Recycling of CO₂ was observed in developing leaves without concomitant net CO₂ uptake. Accumulation of malate was positively correlated with increased cell sap osmolality and improved daily water use efficiency. The importance of flexibility in carbon uptake pathway and of recycling CO₂ for the ecological success of the plant is discussed.     

Growth, CO2 uptake, and responses of the carboxylating enzymes to inorganic carbon in two highly productive CAM species at current and doubled CO2 concentrations

In Agave salmiana Otto ex Salm. var. salmiana grown for 4½ months in open-top chambers, 55% more leaves unfolded and 52% more fresh mass was produced at 730 than at 370μmol CO₂ mol^?1. A doubling of the CO₂ concentration also stimulated growth in another highly productive CAM species, Opuntia ficus-indica (L.) Miller, leading to earlier initial ion and 37% more daughter cladodes. Substantial net CO₂ uptake occurred earlier in the afternoon and lasted longer through the night for A. salmiana at 730 than at 370μmol CO₂ mol^?1, resulting in 59% more total daily net CO₂ uptake. The Michaelis constant (HCO₃^?) for PEPCasc was 15% lower for A. salmiana and 44% lower for O. ficus-indica when the CO₂ concentration was doubled; the percentage of Rubisco in the activated state in vivo was on average 64% higher at the doubled CO₂ concentration. Thus the substantial increases in net CO₂ uptake and biomass production that occurred in these two CAM species when the ambient CO₂ concentration was doubled resulted mainly from higher inorganic carbon levels for their carboxylating enzymes, a greater substrate affinity for PEPCase, and a greater percentage of Rubisco in the activated state.     

CAM induction by photoperiodism in green callus cultures from a CAM plant

Abstract Green calli obtained from leaves of the CAM-inducible plant Kalanchoe blossfeldiana cv. Montezuma were grown either under long-day or short-day regimes in the Phytotron of Gif-sur-Yvette. Callus cells were found to be CAM inducible by the short-day treatment, according to levels and diurnal oscillations of malate pools and phosphor-enolpyruvate (PEP) carboxylase (EC.4.1.1.31) capacity. De novo synthesis of PEP carboxylase was shown to occur under the short-day regime. In spite of continuous net CO² output, CAM-like patterns of CO₂ exchange by calli were obtained under the short-day treatment. After 2 months under both photoperiodic conditions, spontaneous organogenesis in callus tissues gave rise to numerous shootlets which were determined as photoperiod dependent for CAM; the same was so for plants originating from these shootlets.     

Differential effects of drought and light levels on accumulation of citric and malic acids during CAM in Clusia

Night-time citrate accumulation has been proposed as a response to stress in CAM plants. To address this hypothesis, gas exchange patterns and nocturnal acid accumulation in three species of Clusia were investigated under controlled conditions with regard to water stress and responses to low and high photosynthetic photon flux density (PPFD). Under high PPFD, leaves of Clusia nocturnally accumulated large amounts of both malic and citric acids. Under low PPFD and well-watered conditions, substantial night-time citrate accumulation persisted, whereas malate accumulation was close to zero. Malate accumulation and night-time CO₂ uptake from the atmosphere declined in all three species during prolonged drought periods, whereas citrate accumulation remained similar or increased. Recycling of respiratory CO₂ was substantial for both well-watered and water-stressed plants. The suggestion that citrate accumulation is energetically more favourable than malate accumulation is not supported if the source of CO₂ for the formation of malate is respiratory CO₂. However, the breakdown of citric acid to pyruvate in the light period releases three molecules of CO₂, while the breakdown of malic acid releases only one CO₂ per pyruvate formed. Thus, citric acid should be more effective than malic acid as a mechanism to increase CO₂ concentration in the mesophyll and may help to prevent photoinhibition. Organic acid accumulation also affected the vacuolar pH, which reached values of 2·6–3·0 at dawn. At these pH values, the transport of 2H⁺/ATP is still feasible, suggesting that it is the divalent form of citrate which is being transported in the vacuoles. Since citrate is a well-known buffer, and Clusia spp. show the largest day-night changes in organic acid levels measured in any CAM plant, it is possible that citrate increases the buffer capacity of the vacuoles. Indeed, malate and titratable acidity levels are positively related to citrate levels. Moreover, Clusia species that show the highest nocturnal accumulation of organic acids are also the ones that show the greatest changes in citric acid levels.     

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

The carbon balance of shade-grown Ananas comosus was investigatedwith regard to nitrogen supply and responses to high PAR. Netdark CO₂ uptake was reduced from 61.2 to 38.5 mmol CO₂ m^–2in N limited (–N) plants grown under low PAR (60 µmolm^–2 s^–1) and apparent photon yield declined from0.066 to 0.034 (mol 0².mol^–1 photon), although photosyntheticcapacities (measured under 5% CO₂) were similar. Following transferfor 7 d to high PAR (600. µmol m^–2 s^–1), netCO² uptake at night increased by 14% in +N plants, and daytimephotosynthetic capacity was higher, with a maximum value of7.8 µmol m^–2 s^–1. The magnitude of dark CO₂ fixation during CAM was measured asdawn—dusk variations in leaf-sap titratable acidity (H+)and as the proportion of malic and citric acids. The contributionfrom re-fixation of respiratory CO₂ recycling (measured as thedifference between net CO₂ uptake and malic acid accumulation)varied with growth conditions, although it was generally lower(30%) than reported for other bromeliads. Assuming a stoichiometryof 2H+: malate and 3H+: citrate, there was a good agreementbetween titratable protons and enzymatically determined organicacids. The accumulation of citric acid was related to nitrogensupply and PAR regime, increasing from 7.0 mol m–3 (+Nplants) to 18 mol m^–3 (–N plants) when plants weretransferred to high PAR; malate: citrate ratios decreased from13.1 to 2.5 under these conditions. Under the low PAR regime, leaf-sap osmotic pressure increasedat night in proportion to malic acid accumulation. However,following the transfer to high PAR for 7 d, there was a muchgreater depletion of soluble sugars at night which correspondedto a decrease in leaf-sap osmotic pressure. Although a rolefor citric acid in CAM has not been properly defined, it appearsthat the accepted stoichiometry for CAM in terms of gas exchange,titratable acidity, malic acid and osmotic pressure may nothold for plants which accumulate citric acid. Key words: Ananas comosus, CAM, citric acid accumulation, carbon recycling     

Crassulacean acid metabolism (CAM) in an epiphytic ant-plant, Myrmecodia beccarii Hook.f. (Rubiaceae)

This study demonstrates unequivocally the presence of crassulacean acid metabolism (CAM) in a species of the Rubiaceae, the fourth largest angiosperm plant family. The tropical Australian endemic epiphytic ant-plant, Myrmecodia beccarii Hook.f., exhibits net CO₂ uptake in the dark and a concomitant accumulation of titratable acidity in plants in the field and in cultivation. Plants growing near Cardwell, in a north Queensland coastal seasonally dry forest of Melaleuca viridiflora Sol. ex Gaertn., accumulated ~50 % of their 24 h carbon gain in the dark during the warm wet season. During the transition from the wet season to the dry season, 24 h carbon gain was reduced whilst the proportion of carbon accumulated during the dark increased. By mid dry season many plants exhibited zero net carbon uptake over 24 h, but CO₂ uptake in the dark was observed in some plants following localised rainfall. In a shade-house experiment, droughted plants in which CO₂ uptake in the light was absent and dark CO₂ uptake was reduced, were able to return to relatively high rates of CO₂ uptake in the light and dark within 12 h of rewatering.     

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.