Photoinhibition of the CAM succulent Opuntia basilaris growing in Death Valley: evidence from 77K fluorescence and quantum yield

Summary Diurnal measurements of low temperature (77K) fluorescence at 690 nm (PS II) from north, south, east, and west facing cladode surfaces of Opuntia basilaris in Death Valley, California were made on six occasions during 1985. The absolute levels of F _o(instantaneous fluorescence) and F _m(maximum fluorescence), as well as the ratio F _v/F _m(variable fluorescence, F _m-F _o, over maximum fluorescence), were greater in the north face relative to the other faces. Diurnal decreases in F _o, F _mand F _v/F _mwere found concomitant with increases in incident photon flux area density (PFD). F _v/F _mwas fairly low throughout the year, indicative of photoinhibition, but became somewhat elevated after a spring rain. In early fall the quantum yield of the south face was considerably depressed relative to that of the north face, and corresponding differences were observed in F _v/F _m. A decrease in PFD during growth of glasshouse plants led to an increase in chlorophyll concentration, F _oand F _m, but not F _v/F _m. Although there was some variability in the quantum yield of well watered glasshouse cladodes, a correlation was found between quantum yield and the light and CO₂ saturated rate of photosynthesis. When O. basilaris was water stressed under glasshouse conditions, reductions in quantum yield, F _m, and F _v/F _mwere observed. Reductions in F _v/F _malways indicated a reduced quantum yield, although the converse was not necessarily so in well watered glasshouse plants. The results of this study indicate that O. basilaris is likely to experience photoinhibition throughout much of its life in Death Valley.Abbreviations CAM crassulacean acid metabolism - MPa megapascal - PFD photon flux area density - PS II photosystem II - vater potential - F _o instantaneous fluorescence - F _m maximum fluoescence - F _o variable fluorescence     

Photosynthetic flexibility and ecophysiological plasticity: questions and lessons from Clusia, the only CAM tree, in the neotropics

The discovery of crassulacean acid metabolism (CAM) in the trees of Clusia: arrival in the limelight of international research 8 II. Phylogeny 8 III. Photosynthetic physiotypes 10 IV. Metabolic flexibility: organic acid variations 12 V. The environmental control of photosynthetic flexibility 13 VI. Phenotypic plasticity: physiotypes and morphotypes 16 VII. Ecological amplitude and habitat impact 16 VIII. Conclusions and outlook 21 Acknowledgements 22 References 22 Summary It is the aim of this review to present a monographic survey of the neotropical genus Clusia on scaling levels from molecular phylogeny, metabolism, photosynthesis and autecological environmental responses to ecological amplitude and synecological habitat impact. Clusia is the only dicotyledonous genus with real trees performing crassulacean acid metabolism (CAM). By way of introduction, a brief historical reminiscence describes the discovery of CAM in Clusia and the consequent increase in interest in studying this particular genus of tropical shrubs and trees. The molecular phylogeny of CAM in the genus is compared with that in Kalancho? and the Bromeliaceae. At the level of metabolism and photosynthesis, the great plasticity of expression of photosynthetic physiotypes, i.e. (i) C(3) photosynthesis, (ii) CAM including CAM idling, (iii) CAM cycling and (iv) C(3)/CAM-intermediate behaviour, as well as metabolic flexibility in Clusia is illustrated. At the level of autecology, the factors water, irradiance and temperature, which control photosynthetic flexibility, are assessed. The phenotypic plasticity of physiotypes and morphotypes is described. At the level of synecology, the ecological amplitude of Clusia in the tropics and the relations to habitat are surveyed.     

Shifts in the optimal temperature for nocturnal CO2 uptake caused by changes in growth temperature for cacti and agaves

When the day/night air temperatures were raised from 10°C/10°C to 30°C/30°C, the optimal tempearture for nocturnal CO² uptake by six species of cacti and three species of agave shifted from an average of 12°C to an average of 20°C. The maximum rate of CO² uptake was higher for Agave americana at the higher ambient temperature, lower for A. deserti , and much lower for A. utahensis , consistent with the relative mean temperatures of their native habitats. For the cactus Coryphantha vivipara , which had the greatest temperature shift observed (13°C), the halftime was 8 days for the upward shift and 4 days for the downward shift. The halftimes for the comparable shifts averaged 1.6 days for three other species of cacti and less than 1 day for two agave species. The shifts in the optimal temperature for nocturnal CO² uptake were in response to changes in nighttime temperature, at least for C. vivipara , and reflected temperature responses of both the stomates and the chlorenchyma.     

Photosynthesis in enzymatically isolated leaf cells from the CAM plant Sedum telephium L.

A technique has been developed for the enzymatic isolation of leaf cells from the Crassulacean acid-metabolism plant Sedum telephium. The cells exhibited high activity in both ¹⁴CO₂ incorporation (30–70 mol CO₂ mg^-1 chlorophyll h^-1) and O₂ evolution in the presence of bicarbonate (60–110 mol O₂ mg^-1 chlorophyll h^-1). Half-maximum saturation of ¹⁴CO₂ incorporation occurred at a bicarbonate concentration of ca. 2 mM (20 M CO₂) at pH 8.4 and 30°C. Two types of light-dependent O₂ evolution are reported: O₂ evolution in the absence of exogenously supplied bicarbonate (endogenous O₂ evolution), and bicarbonate-stimulated O₂ evolution. Oxygen evolution in the presence of approximately ambient concentrations of CO₂ appeared to be a combination of the endogenous O₂ evolution and O₂ evolution from fixation of the exogenously supplied CO₂.Abbreviations CAM Crassulacean acid metabolism - cirlo chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PEP phosphoenolpyruvate - RuDP ribulose-1,5-diphosphate     

Expression of P-Enolpyruvate Carboxylase and other Aspects of CAM during the Development of Peperomia camptotricha Leaves

During the development of Peperomia camptotricha leaves, metabolism changes from C₃-photosynthesis to Crassulacean acid metabolism (CAM). The youngest leaves showed no diurnal fluctuation of organic acids or P-enolpyruvate carboxylase (PEPc) activity. There was little evidence for PEPc protein using PEPc antibodies prepared from the CAM form of PEPc, nor was there evidence for PEPc mRNA when tested using a cDNA probe made from CAM P. scandens. As leaves matured, there was a parallel increase in titratable acidity, PEPc activity, PEPc protein, and PEPc mRNA. In leaf whorls 1 through 6, there was a significant linear correlation between the diurnal fluctuation of organic acids and PEPc activity indicating a functional relationship. The specific activity of PEPc increased as leaves matured and the K_m (PEP) decreased indicating that the enzyme was becoming more active. The ratio of PEPc protein to PEPc mRNA decreased as leaves matured. During the expression of CAM, the spongy mesophyll where most of the CAM activity occurs increased in thickness and per cent air space, whereas the palisade mesophyll where most of the C₃ activity occurs did not increase in size dramatically. The diurnal fluctuation of organic acids and the expression of PEPc activity, protein, and mRNA increased as the thickness of the spongy mesophyll increased. During the expression of CAM in Peperomia camptotricha, there appears to be coordinated expression of PEPc mRNA, protein, and activity, the commencement of diurnal organic acid fluctuation, and the development of the CAM-like spongy mesophyll. Thus the evidence suggests that CAM in this species is expressed during normal development and not in response to environmental signals.     

Photosynthetic flexibility in Pedilanthus tithymaloides poit,a CAM plant

The induction of CAM in Pedilanthus tithymaloides (Euphorbiaceae) under water-limited conditions was evaluated by following diurnal oscillations of CO2 fixation, titratable acidity and malic acid content in the leaf extracts. CAM induction was assessed by measuring the activities of phosphoenolpyruvate carboxylase (PEPC), NADH-malate dehydrogenase (MDH) and phosphoenolpyruvate caroxykinase (PEPCK) in the leaves as well. Drought resulted in large increases in the nocturnal acid accumulation and rates of CO2 uptake in the leaves of P. tithymaloides. The drought-induced CAM activity tended to be reversible after re-watering. Nevertheless, under well-watered conditions, plants of P. tithymaloides showed day time CO2 uptake patterns with less pronounced diurnal oscillations of organic acids. Our data indicate that although P. tithymaloides is a CAM plant, environmental variables like drought induce photosynthetic flexibility in this species. This type of plasticity in CAM and metabolic versatility in P. tithymaloides should be an adaptation for prolonged survival under natural adverse edaphic and microclimate situations.     

Activity of enzymes of carbon metabolism during the induction of Crassulacean acid metabolism in Mesembryanthemum crystallinum L.

The maximum extractable activities of twenty-one photosynthetic and glycolytic enzymes were measured in mature leaves of Mesembryanthemum crystallinum plants, grown under a 12 h light 12 h dark photoperiod, exhibiting photosynthetic characteristics of either a C₃ or a Crassulacean acid metabolism (CAM) plant. Following the change from C₃ photosynthesis to CAM in response to an increase in the salinity of in the rooting medium from 100 mM to 400 mM NaCl, the activity of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) increased about 45-fold and the activities of NADP malic enzyme (EC 1.1.1.40) and NAD malic enzyme (EC 1.1.1.38) increased about 4- to 10-fold. Pyruvate, Pi dikinase (EC 2.7.9.1) was not detected in the non-CAM tissue but was present in the CAM tissue; PEP carboxykinase (EC 4.1.1.32) was detected in neither tissue. The induction of CAM was also accompanied by large increases in the activities of the glycolytic enzymes enolase (EC 4.2.1.11), phosphoglyceromutase (EC 2.7.5.3), phosphoglycerate kinase (EC 2.7.2.3), NAD glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), and glucosephosphate isomerase (EC 2.6.1.2). There were 1.5- to 2-fold increases in the activities of NAD malate dehydrogenase (EC 1.1.1.37), alanine and aspartate aminotransferases (EC 2.6.1.2 and 2.6.1.1 respectively) and NADP glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13). The activities of ribulose-1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39), fructose-1,6-bisphosphatase (EC 3.1.3.11), phosphofructokinase (EC 2.7.1.11), hexokinase (EC 2.7.1.2) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) remained relatively constant. NADP malate dehydrogenase (EC 1.1.1.82) activity exhibited two pH optima in the non-CAM tissue, one at pH 6.0 and a second at pH 8.0. The activity at pH 8.0 increased as CAM was induced. With the exceptions of hexokinase and glucose-6-phosphate dehydrogenase, the activities of all enzymes examined in extracts from M. crystallinum exhibiting CAM were equal to, or greater than, those required to sustain the maximum rates of carbon flow during acidification and deacidification observed in vivo. There was no day-night variation in the maximum extractable activities of phosphoenolpyruvate carboxylase, NADP malic enzyme, NAD malic enzyme, fructose-1,6-bisphosphatase and NADP malate dehydrogenase in leaves of M. crystallinum undergoing CAM.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - RuBP ribulose-1,5-bisphosphate     

Comparative Study of the C3/CAM Intermediate Species Clusia parviflora Saldanha et Engi. and the Obligate CAM Species Clusia hilariana Schlecht. Growing Sympatrically Exposed and Shaded in the Coastal Restinga of Brazil

Abstract: The C₃/CAM intermediate species, C/usia parviflora Saldanha et EngI., and the obligate CAM species Clusia hilariana Schlecht., occur sympatrically in the coastal sand dune vegetation of the Restinga of Brazil. Their photosynthetic activity at an exposed and at a shaded site was compared by measuring gas exchange (porometry), chlorophyll a fluorescence parameters, organic acid levels (malic and citric) and carbon isotope ratios. At the shaded site, low photosynthetic photon flux densities (PPFD) strongly restricted photosynthetic activity. However, C parviflora could readily make use of light flecks. At the exposed site, C. parviflora was much less affected by photoinhibition than C. hilariana . The CAM species showed higher apparent rates of linear photosynthetic electron transport (ETR) and higher effective quantum yield of PSII (ΔF/F'_m) than did C. parviflora during high insolation in the middle of the day, i.e., the time of Phase Ill of CAM. Nevertheless, it suffered much more severe acute photoinhibition that was not reversible after 20 min of darkening during this time, and even some chronic photoinhibition not reversible overnight. Comparative studies of sympatric physiotypes with different modes of photosynthesis of a given leaf morphotype, as available in the genus Cksia , challenge some CAM dogmas, e.g., CAM may not always be superior at exposed sites and may not always provide better photoprotection at high PPFD. However, the idea that C₃/CAM plasticity allows occupation of a wider range of habitats is supported.     

Ecophysiological studies on orchids of Madagascar: incidence and plasticity of crassulacean acid metabolism in species of the genus Angraecum Bory

The present study is an investigation on photosynthetic options in an orchid taxon and deals with the mainly epiphytes comprising genus Angraecum Bory. The incidence of crassulacean acid metabolism (CAM) in Angraecum species collected at various habitats in Madagascar was surveyed by analysis of stable carbon isotope composition (¹³C values). The values showed both inter- and intraspecific variability and suggest that in situ about 50% of the analysed species perform C3 photosynthesis, 20% moderate CAM (fixation of external CO₂ during day-and night-time) and 30% pronounced CAM (CO₂ uptake entirely during the night). The photosynthetic behaviour of the species indicated by the ¹³C values was clearly related to the habitat from where the samples derived. In A. eburneum, A. sororium and particularly in A. sesquipedale the stable carbon isotope analysis was complemented by measurements of CAM performance under controlled conditions. The experiments with A. sesquipedale revealed that drought and temperature are important factors modulating CAM, whereas variation of the leaf-to-air water vapor pressure difference was less effective. Altogether, the results of the study support the view that the high biological adaptability and thus the ecological success of the genus Angraecum is largely based on genotypic diversity and intraspecific plasticity of the photosynthetic behaviour.     

Expression of crassulacean acid metabolism in <Emphasis Type="Italic">Clusia hilariana</Emphasis> Schlechtendal in different stages of development in the field

Expression of crassulacean acid metabolism (CAM) in the obligate CAM-tree Clusia hilariana SCHLTDL. was studied in the restinga of Jurubatiba National Park, on the Atlantic coast of Rio de Janeiro state, Brazil, comparing plants at different developmental stages. Between young and mature plants there were trends of differences in six parameters, which are all related to CAM expression. From young to mature plants there were tendencies for a decrease of (1) the degree of succulence, (2) the degree of day/night changes of malic acid levels, (3) titratable acidity with nocturnal acid accumulation, (4) the degree of day/night changes of free hexoses with nocturnal break down, (5) effective quantum use efficiency of photosystem II at high photosynthetic photon flux density, and (6) protection from photoinhibition. These tendencies form a clear pattern which suggests that CAM was somewhat more pronounced in leaves of young plants than in leaves of mature plants. A developmental regulation may be involved. However, the observations are probably best explained by stress, since in the dry soils of the restinga young plants have no access to the ground water table while adult trees develop extensive root systems.     

Comparative measurements of chlorophyll a fluorescence, acid accumulation and gas exchange in exposed and shaded plants of Clusia minor L. and Clusia multiflora H. B. K. in the field

 Changes in chlorophyll a fluorescence during the day and diurnal-changes of net CO₂-exchange and organic acid contents were determined in two species of the genus Clusia during the dry season in Venezuela. The investigations included plants of the C₃/CAM intermediate species Clusia minor and the C₃ species C. multiflora growing at exposed and shaded sites. Both species showed a C₃ pattern of net CO₂-exchange at the exposed site. In the shade under extreme drought stress C. minor showed a weak expression of CAM without CO₂-uptake during the afternoon (phase IV of CAM). C. multiflora growing in the shade exhibited a C₃-pattern of net CO₂-exchange and a small but significant nocturnal accumulation of citrate. Shaded plants of C. minor were able to double their light utilisation for electron transport and to reduce non-photochemical quenching during phase III compared to phase II of CAM. Furthermore, increase of electron transport rate through photosystem II in phase III of CAM is correlated to decarboxylation of malate. At the exposed site C. multiflora was less negatively affected by high PPFD than C. minor. This was shown by a lower reduction of potential electron quantum yield (F_v/F_m) and higher light utilisation of electron transport of C. multiflora compared to C. minor. At the exposed site C. minor did not make use of the CAM option to increase light utilisation of electron transport and to reduce non-photochemical quenching as did the plants growing in the shade. Received: 20 March 1996 / Accepted: 24 June 1996     

Diurnal changes in photochemical efficiency,the reduction state of Q,radiationless energy dissipation,and non-photochemical fluorescence quenching in cacti exposed to natural sunlight in northern Venezuela

Summary Diurnal measurements of chlorophyll a fluorescence from cacti (Nopalea cochenillifera, Opuntia ficus-indica, and Opuntia wentiana) growing in northern Venezuela were used to determine photochemical fluorescence quenching related to the reduction state of the primary electron acceptor of PS II as well as non-photochemical fluorescence quenching which reflects the fraction of energy going primarily into radiationless deexcitation. The cladodes used in this study were oriented such that one surface received direct sunlight in the morning and the other one during the afternoon. Both surfaces exhibited large increases in radiationless energy dissipation from the photochemical system accompanied by decreases in PS II photochemical efficiency during direct exposure to natural sunlight. During exposure to sunlight in the morning, dissipation of absorbed light energy through photosynthesis and radiationless energy dissipation was sufficient to maintain Q, the primary electron acceptor for PS II, in a low reduction state. During exposure to sunlight in the afternoon, however, the reduction state of Q rose to levels greater than 50%, presumably due to a decrease in photosynthetic electron transport as the decarboxylation of the nocturnally accumulated malic acid was completed. Exposure to direct sunlight in the afternoon also led to more sustained increases in radiationless energy dissipation. Furthermore, the increases in radiationless energy dissipation during exposure of a water-stressed cladode of O. wentiana to direct sunlight were much greater than those from other well-watered cacti, presumably due to sustained stomatal closure and decreased rates of photosynthetic electron transport. These results indicate that the radiationless dissipation of absorbed light is an important process in these CAM plants under natural conditions, and may reflect a protective mechanism against the potentially damaging effects of the accumulation of excessive energy, particularly under conditions where CO₂ availability is restricted.Abbreviations CAM crassulacean acid metabolism - F _o instantaneous fluorescence emission - F _M maximum fluorescence emission - F _v variable fluorescence emission - K _D rate constant for radiationless energy dissipation in the antenna chlorophyll - PFD photon flux density - PS I photosystem I - PS II photosystem II - Q primary electron acceptor of photosystem II - q _NP non-photochemical fluorescence quenching - q _P photochemical fluorescence quenching - T _C cladode temperature     

A Comparative Study by δ13C-Analysis of Crassulacean Acid Metabolism (CAM) in Kalanchoë (Crassulaceae) Species of Africa and Madagascar

The carbon isotope ratios (δ¹³C values) of samples of Kalanchoë species collected in Africa were compared with previous data obtained with species from Madagascar. In contrast to the Malagasy species which cover the whole range of δ¹³C values from ?10 to ? 30%_o, indicating high inter- and intraspecific diversity of CAM performance, in the African species nearly all δ¹³C values were less negative than ?18%₀. Thus, in the African species the CAM behaviour is characterized by CO₂ uptake proceeding mainly during the night. The distribution of δ¹³C values among the species clearly mirrors the taxonomic groups and the three sections of the genus Kalanchoë sensu lato. The Kitchingia section comprises only groups having CAM with a high proportion of carbon acquisition by the C₃-pathway of photosynthesis. The same holds true for the first three groups of the Bryophyllum section, whereas in the following groups of the section CAM with CO₂ proceeding mainly during the night is common. The latter CAM mode is typical also for the majority of groups and species in the section Eukalanchoë. The African Kalanchoë species belong to the Eukalanchoë section, whereas in Madagascar all three sections are abundant. The data support the view that the centre of adaptive radiation of the genus is located in Madagascar. They also suggest that high CAM variability is abundant in the more primitive taxa of the genus, whereas the phylogenetically more derived taxa show a stereotype CAM with CO₂ uptake taking place only during the night.     

Patterns of gas exchange and organic acid oscillations in tropical trees of the genus Clusia

Summary Gas exchange patterns and nocturnal acid accumulation were examined in four species of Clusia under simulated field conditions in the laboratory. Clusia alata and C. major had midday stomatal closure, substantial net CO₂ exchange ( ) during the night, and the highest water use efficiency (WUE). C. venosa showed a pattern similar to a C₃ plant, with nighttime stomatal closure, while C. minor maintained positive continuously throughout a 24-h period. However, large changes in titratable acidity, which closely matched changes in citrate and malate levels, indicated that Crassulacean acid metabolism (CAM) is active in all four species. C. venosa showed dawn-dusk oscillations in titratable acidity that were higher than the values reported for other C₃-CAM intermediates, while the nighttime acid accumulation of 998 mol m^–3 observed in C. major is unsurpassed by any other CAM plant. Moreover, the dawn-dusk changes in citrate levels of over 65 mol m^–3 in C. alata and C. minor, and over 120 mol m^–3 in C. major, are 3–6 times higher than values reported for other CAM plants. Although these oscillations in citrate levels were quite large, and the nighttime dark respiration rates were high, the O₂ budget analysis suggestes that only part of the reducing power generated by the synthesis of citric acid enters the respiratory chain. Dawn-dusk changes in malate levels were just over 50 mol m^–3 for C. venosa but over 300 mol m^–3 for C. major. Between 28% (C. major) and 89% (C. venosa) of the malate accumulated during the night was derived from recycled respiratory CO₂. These daily changes in malate and citrate levels also contributed significantly to changes in leaf sap osmolality. This variability in CO₂ uptake patterns, the recycling of nighttime respiratory CO₂, and the high WUE may have contributed to the successful invasion of Clusia into a wide range of habitats in the tropics.     

Comparison of Photosynthetic Responses of the Sympatric Tropical C3 Species Clusia multiflora H. B. K. and the C3-CAM Intermediate Species Clusia minor L. to Irradiance and Drought Stress in a Phytotron1

Abstract: Clusia multiflora H. B. K., an obligate C₃ species and Clusia minor L. a C₃/CAM intermediate species, are two physio-types of a similar morphotype. They can sympatrically occupy secondary savanna sites exposed to high insolation in the tropics. In C. multiflora severe stress, i.e., switching shade-grown plants to high light plus drought, resulted in leaves browning or yellowing and becoming necrotic. However, in long-term light stress C. multiflora was able to grow new leaves with their photosynthetic apparatus fit for high light conditions. Shade-grown C. minor readily overcame switching to high light conditions and drought, responding by a rapid change from C₃ photosynthesis to CAM. Decreasing _soil led to increased abscisic acid levels in the leaves of C. minor, however CAM induction was not directly related to this and was mainly determined by increased PPFD. Both species were capable of rapid accumulation of zea-xanthin for acute photoprotection following high PPFD exposure. The maximum capacity for zeaxanthin accumulation was larger in C. minor, but under steady high PPFD it only partially made use of this capacity, relying on high internal CO₂ concentrations of Phase Ill of CAM, in addition to zeaxanthin, for acute photo-protection. Thus, by different means the two species perform well under high light conditions. However, C. multiflora needs time for development of adapted leaves under such stress conditions while the more flexible C. minor can readily switch from low light to high light conditions.     

Antioxidative protection in the inducible CAM plant Sedum album L. following the imposition of severe water stress and recovery

The antioxidative protection during the C₃-CAM shift induced by water stress was investigated in the temperate succulent Sedum album L. The C₃-CAM shift was characterized in terms of CO₂ exchange, titratable acidity and phosphoenolpyruvate carboxylase activity. Well-watered plants displayed C₃-like patterns of gas exchange and exhibited a mild day-night acid fluctuation indicating that those plants were performing CAM-cycling metabolism. Imposed drought highly stimulated CAM cycling, decreasing the net CO₂ uptake during the day, eliminating net CO₂ efflux at night and stimulating tissue acid fluctuations. As water deficit developed, chlorophyll fluorescence measurements showed a decrease in the Fv/Fm ratio, indicating that photoinhibition could follow after severe drought. Protection might be performed by the increased activity of enzymes involved in the destruction of free radicals and oxidants, but their response depended on the water status of the plant. Ascorbate peroxidase and superoxide dismutase activities increased in plants subjected to mild stress but declined during severe water stress. Catalase activity, however, was quite stable under mild water stress and was clearly inhibited under severe water stress. At this stage, glutathione reductase and monodehydroascorbate reductase seemed to be very important in the protection against oxidants, both increasing considerably their activities under severe water stress. Even if recycling has been shown to alleviate photoinhibition, our results clearly demonstrate that antioxidative enzymes play an important role in the protection of plants from oxidants during the C₃-CAM shift induced by water stress.     

Superoxide dismutase activity in callus from the C3-CAM intermediate plant Mesembryanthemum crystallinum

In light-grown callus obtained from M. crystallinum hypocotyls, three classes of superoxide dismutase (SOD): Mn-, Fe- and Cu/ZnSOD were identified. Callus cultured on a medium containing 0.4 M NaCl showed an increase in FeSOD activity on day 4 of the experiment. In contrast, Cu/ZnSOD activity was higher over 16 days of the experiment. Salinity stress induces oxidative stress mainly for the cytosolic SOD form (Cu/ZnSOD). After 16 days of callus culture on salt-containing medium, diurnal malate oscillations, and an increase in NADP-malic enzyme activity were noticed. These results strongly suggest that C₃-CAM transition can also be expressed at the cellular level. Therefore, callus tissue could be a useful model, similar to a whole plant, for investigation of mechanisms of stress responses in M. crystallinum.     

The occurrence of crassulacean acid metabolism in Cymbidium (Orchidaceae) and its ecological and evolutionary implications

Crassulacean acid metabolism (CAM) is one of the photosynthetic pathways regarded as adaptations to water stress in land plants. Little is known about correlations among the level of CAM activity, environment of habitat, life form, and phylogenetic relationship of a plant group from an evolutionary perspective. We examined these relationships in 18 species of Cymbidium (Orchidaceae) because the genus shows distinctive diversification of habitats and life forms. The photosynthetic type was classed into three categories, strong CAM, weak CAM, and C₃ on the basis of CAM activity. CAM expression in Cymbidium was confined to the epiphytic and lithophytic species. Especially, all of these species from tropical to subtropical rainforest exhibited CAM activity. On the other hand, the terrestrial species always exhibited C₃ metabolism irrespective of their varied habitats. Regarding the evolution of photosynthetic characters, weak CAM was the ancestral state in Cymbidium and strong CAM and C₃ metabolism occurred subsequently. The evolution of strong CAM likely enabled Cymbidium to extend to exposed sites in tropical lowland where marked water stress exists. Further, different levels of CAM activity characterized each species and such potential plasticity of CAM may realize the radiation of Cymbidium into sites with different environmental conditions.