Obligate Crassulacean Acid Metabolism Plant Research Articles

Comparative genomics analysis of drought response between obligate CAM and C3 photosynthesis plants

Crassulacean acid metabolism (CAM) plants exhibit elevated drought and heat tolerance compared to C3 and C4 plants through an inverted pattern of day/night stomatal closure and opening for CO2 assimilation. However, the molecular responses to water-deficit conditions remain unclear in obligate CAM species. In this study, we presented genome-wide transcription sequencing analysis using leaf samples of an obligate CAM species Kalanchoë fedtschenkoi under moderate and severe drought treatments at two-time points of dawn (2-h before the start of light period) and dusk (2-h before the dark period). Differentially expressed genes were identified in response to environmental drought stress and a whole genome wide co-expression network was created as well. We found that the expression of CAM-related genes was not regulated by drought stimuli in K. fedtschenkoi. Our comparative analysis revealed that CAM species (K. fedtschenkoi) and C3 species (Arabidopsis thaliana, Populus deltoides ‘WV94’) share some common transcriptional changes in genes involved in multiple biological processes in response to drought stress, including ABA signaling and biosynthesis of secondary metabolites.

Flowering responses under elevated CO2 and graded nutrient supply in Phalaenopsis Queen Beer ‘Mantefon’

Obligate crassulacean acid metabolism (CAM) plants rely heavily on nocturnal CO2 fixation; however, little is known about how changes in CO2 concentration affect the nutrient requirements of these plants. To evaluate the combined effect of CO2 concentration and nutrient provision on obligate CAM plants, we cultivated Phalaenopsis Queen Beer ‘Mantefon’ under two CO2 concentrations (400 or 800 μmol· mol−1) and with four nutrient solutions (EC [electrical conductivity] of 1.0, 2.0, 3.0, or 4.0 dS·m−1) for 17 weeks. Various flowering characteristics of Phalaenopsis (flower spike length, the number of lateral branches, floral buds, and flowers, days to flowering, flower size, and flower color) were then investigated during the reproductive stage. Overall, higher CO2 concentrations enhanced the growth and productivity of Phalaenopsis. More specifically, flower spike lengths were longer and more lateral branches were initiated under 800 μmol·mol−1 CO2 compared to plants cultivated under 400 μmol·mol−1 CO2 with an EC of 3.0 or 4.0 dS·m−1. Additionally, plants cultivated under 800 μmol·mol−1 CO2 had greater number of floral buds and flowers than those cultivated under 400 μmol·mol−1 CO2, regardless of EC. Floral buds and flowers were not linearly increased by increasing EC, regardless of CO2 concentration. Flowering time was faster in plants cultivated under 800 μmol·mol−1 CO2. Altogether, these results suggest that an elevated atmospheric CO2 concentration positively affects the flower spike length and the number of lateral branches, floral buds, and flowers of Phalaenopsis and also indicate that nutrient levels can be adjusted to improve the growth of obligate CAM plants.

Enhancing Abiotic Stress Response via a Kalanchoe fedtschenkoi NF‐Y Transcription Factor in C3 Plants

Global crop production is expected to decrease because of increasing abiotic stressors including extremes in temperature, water, light intensity, nutrients, and radiation due to climate change and anthropogenic activities. Increased abiotic stressors will alter the metabolism in C3 plants and thus negatively affect their growth and productivity. Developing C3 crop varieties with improved abiotic stress tolerances emerges as the most sustainable solution towards ensuring global food security under the predicted climate change scenario. Crassulacean Acid Metabolism (CAM) plants are considered well‐adapted to drought, heat, increased light intensities, higher UV irradiation, and are far more water‐use efficient compared to C3 photosynthesis plants. The use of abiotic stress‐responsive transcription factors (TFs) from CAM plants is one feasible approach to improve crop production under adverse abiotic stress conditions. We have identified a NF‐YB TF from the heat‐tolerant obligate CAM plant Kalanchoe fedtschenkoi as a possible regulator of abiotic stresses. This NF‐Y is a ubiquitous transcription factor that is expressed as one of three possible isoforms NF‐YA, NF‐YB, or NF‐YC. Their isoforms interact to form a heterotrimeric complex with each subunit playing key roles in the abiotic stress responses including drought, heat, freezing, and salinity. We are functionally characterizing this KfNF‐YB via overexpression in Arabidopsis thaliana to determine its role in abiotic stress responses. Preliminary results following in vitro salinity stress assay of T2 transgenic lines after 12 days in MS media with 200 mM NaCl indicate increased root growth, whereas the wild type had retarded root growth. In addition, transgenic lines had higher biomass dry weight compared to wild type after 12 days. T2 lines also have increased rosette size in two‐week old transgenic A. thaliana compared to wild type. We will discuss the implications of the preliminary results as a potential strategy to enhance multiple abiotic stress tolerance responses in C3 plants.Support or Funding InformationThis work is funded by South Dakota Board of Regents Grant #A18‐0015‐001 to BWM Wone and supported in part by U. S. Department of Energy, Office of Science, Genomic Science Program under award #DE‐SC0008834 to JC Cushman.

Agrobacterium–mediated Transformation of Kalanchoe laxiflora

Plants with crassulacean acid metabolism (CAM) generally utilize water 20%–80% more efficiently than non-CAM plants. The whole genomes of several CAM plants have been sequenced or are being sequenced. For effective genome characterization and genome editing of CAM plants, an efficient transformation system is essential. In this study, we developed an Agrobacterium-mediated transformation protocol for Kalanchoe laxiflora, an obligate CAM plant,by optimizing several factors affecting the transformation efficiency. Agrobacterium strains AGL1, C58, EHA105, and GV3101 were all suitable for K. laxiflora transformation. Fifty-nine percent of the leaf explants yielded kanamycin-resistant and GUS-positive shoots. Polymerase chain reaction and quantitative real-time PCR (qRT-PCR) using gusA-, gusPlus-, nptII- and hpt-specific primers confirmed that the transgenes were integrated into K. laxiflora genome and expressed. This efficient transformation system will allow effective functional characterization of genes through over- or down-expression, knockout, or genome editing.

A photosynthesis continuous monitoring system for CAM plants

Diurnal CO2 exchanges in crassulacean acid metabolism (CAM) plants are significantly different from those in C3 and C4 plants. The instantaneous short-time CO2 exchange of a single leaf measured by commercial portable photosynthesis measuring systems with a small leaf chamber cannot reflect the plant photosynthetic capacity for CAM plants because of the CO2 fixation property. Therefore, a photosynthesis continuous monitoring system with two canopy cuvettes was developed for measuring diurnal net CO2 exchange rates for CAM plants. To evaluate stability and applicability of the photosynthesis continuous monitoring system, continuous measurement of net CO2 exchange rates of plants with different photosynthetic pathways were conducted. An obligate CAM plant (Kalanchoe daigremontiana), four facultative CAM plants (Dendrobium officinale, D. chrysotoxum, D. nobile, and D. primulinum), a C3 plant (Strawberry, Fragaria ananassa), and a C4 plant (Corn, Zea mays) were selected as model plants. K. daigremontiana had a significant CO2 absorption during the dark period and its net CO2 exchange rates fluctuated around 0 μmol/(m2•s) during the photoperiod in a growth chamber. Net CO2 exchange rates of F. ananassa and Z. mays in a greenhouse gradually increased after sunrise, reaching a maximum at about 12:00, and then gradually decreased to negative values during the night time. It is interesting to observe that D. officinale in the greenhouse and growth chamber absorbed CO2 during both day and night times. The photosynthetic pathways of D. chrysotoxum, D. nobile, and D. primulinum were also well distinguished by this photosynthesis continuous monitoring system. The results showed that the photosynthesis continuous monitoring system is capable for quantitative evaluation of diurnal net CO2 exchange characteristics not only in the CAM plants but also in small size C3 and C4 plants with low net photosynthetic rates for long-time and high-accuracy measurements. Keywords: diurnal net CO2 exchanges, infrared CO2 analyzer, mass airflow meter, net photosynthetic rate, photosynthetic pathway DOI: 10.25165/j.ijabe.20191203.4885 Citation: Cheng Y S, He D X. A photosynthesis continuous monitoring system for CAM plants. Int J Agric & Biol Eng, 2019; 12(3): 141–146.

Effect of Light/Dark Cycle on Photosynthetic Pathway Switching and CO2 Absorption in Two Dendrobium Species.

Many Dendrobium species are both ornamental and medicinal plants in China. Several wild species have been exploited to near extinction, and facility cultivation has become an important way to meet the great market demand. Most Dendrobium species have evolved into crassulacean acid metabolism (CAM) pathways in adapting to harsh epiphytic environment, leading to low daily net CO2 absorption. Photosynthetic pathways of many facultative CAM plants are regulated by various environmental factors. Light/dark cycle plays an important role in regulating the photosynthetic pathway of several CAM species. The aims of this study were to investigate whether the photosynthetic pathway of Dendrobium species could be regulated between C3 and CAM by changing light/dark cycles and the daily net CO2 absorption could be enhanced by shortening light/dark cycle. In this study, net CO2 exchange rates of D. officinale and D. primulinum were monitored continuously during two different light/dark cycles conversion compared to Kalanchoe daigremontiana as an obligate CAM plant. The net CO2 exchange pattern and stomatal behavior of D. officinale and D. primulinum were switched from CAM to C3-like by changing the light/dark cycle from 12/12 h to 4/4 h. However, this switching was not completely reversible. Compared to the original 12/12 h light/dark cycle, the dark, light, and daily net CO2 exchange amount of D. officinale were significantly increased after the light/dark cycle was changed from 4/4 h to 12/12 h, but those in D. primulinum was opposite and those in K. daigremontiana was not affected. Daily net CO2 exchange amount of D. officinale increased by 47% after the light/dark cycle was changed from 12/12 h to 4/4 h, due to the sharp increase of light net CO2 exchange amount. However, the large decrease of dark net CO2 exchange amount could not be offset by increased light net CO2 exchange amount, leading to reduced daily net CO2 exchange amount of D. primulinum. In conclusion, the 4/4 h light/dark cycle can induce the photosynthetic pathway of D. officinale and D. primulinum to C3-like, and improve the daily CO2 absorption of D. officinale.

Crassulacean Acid Metabolism Abiotic Stress-Responsive Transcription Factors: a Potential Genetic Engineering Approach for Improving Crop Tolerance to Abiotic Stress.

This perspective paper explores the utilization of abiotic stress-responsive transcription factors (TFs) from crassulacean acid metabolism (CAM) plants to improve abiotic stress tolerance in crop plants. CAM is a specialized type of photosynthetic adaptation that enhances water-use efficiency (WUE) by shifting CO2 uptake to all or part of the nighttime when evaporative water losses are minimal. Recent studies have shown that TF-based genetic engineering could be a useful approach for improving plant abiotic stress tolerance because of the role of TFs as master regulators of clusters of stress-responsive genes. Here, we explore the use of abiotic stress-responsive TFs from CAM plants to improve abiotic stress tolerance and WUE in crops by controlling the expression of gene cohorts that mediate drought-responsive adaptations. Recent research has revealed several TF families including AP2/ERF, MYB, WRKY, NAC, NF-Y, and bZIP that might regulate water-deficit stress responses and CAM in the inducible CAM plant Mesembryanthemum crystallinum under water-deficit stress-induced CAM and in the obligate CAM plant Kalanchoe fedtschenkoi. Overexpression of genes from these families in Arabidopsis thaliana can improve abiotic stress tolerance in A. thaliana in some instances. Therefore, we propose that TF-based genetic engineering with a small number of CAM abiotic stress-responsive TFs will be a promising strategy for improving abiotic stress tolerance and WUE in crop plants in a projected hotter and drier landscape in the 21st-century and beyond.

Photosynthesis continuous monitoring system for CAM plants

Diurnal CO2 exchanges in crassulacean acid metabolism (CAM) plants are significantly different from those in C3 and C4 plants. The instantaneous short-time CO2 exchange of a single leaf measured by commercial portable photosynthesis measuring systems with a small leaf chamber cannot reflect the plant photosynthetic capacity for CAM plants because of the CO2 fixation property. Therefore, a photosynthesis continuous monitoring system with two canopy cuvettes was developed for measuring diurnal net CO2 exchange rates for CAM plants. To evaluate stability and applicability of the photosynthesis continuous monitoring system, continuous measurement of net CO2 exchange rates of plants with different photosynthetic pathways were conducted. An obligate CAM plant (Kalanchoe daigremontiana), four facultative CAM plants (Dendrobium officinale, D. chrysotoxum, D. nobile, and D. primulinum), a C3 plant (Strawberry, Fragaria ananassa), and a C4 plant (Corn, Zea mays) were selected as model plants. K. daigremontiana had a significant CO2 absorption during the dark period and its net CO2 exchange rates fluctuated around 0 μmol/(m2•s) during the photoperiod in a growth chamber. Net CO2 exchange rates of F. ananassa and Z. mays in a greenhouse gradually increased after sunrise, reaching a maximum at about 12:00, and then gradually decreased to negative values during the night time. It is interesting to observe that D. officinale in the greenhouse and growth chamber absorbed CO2 during both day and night times. The photosynthetic pathways of D. chrysotoxum, D. nobile, and D. primulinum were also well distinguished by this photosynthesis continuous monitoring system. The results showed that the photosynthesis continuous monitoring system is capable for quantitative evaluation of diurnal net CO2 exchange characteristics not only in the CAM plants but also in small size C3 and C4 plants with low net photosynthetic rates for long-time and high-accuracy measurements. Keywords: diurnal net CO2 exchanges, infrared CO2 analyzer, mass airflow meter, net photosynthetic rate, photosynthetic pathway DOI: 10.25165/j.ijabe.20191203.4885 Citation: Cheng Y S, He D X. A photosynthesis continuous monitoring system for CAM plants. Int J Agric & Biol Eng, 2019; 12(3): 141–146.

A model of environmental limitations on production of Agave americana L. grown as a biofuel crop in semi-arid regions.

Plants that use crassulacean acid metabolism (CAM) have the potential to meet growing agricultural resource demands using land that is considered unsuitable for many common crop species. Agave americana L., an obligate CAM plant, has potential as an advanced biofuel crop in water-limited regions, and has greater cold tolerance than other high-yielding CAM species, but physiological tolerances have not been completely resolved. We developed a model to estimate the growth responses of A. americana to water input, temperature, and photosynthetically active radiation (PAR). The photosynthetic response to PAR was determined experimentally by measuring the integrated leaf gas exchange over 24 h after acclimation to six light levels. Maximum CO2 fixation rates were observed at a PAR intensity of 1250 µmol photons m-2 s-1. Growth responses of A. americana to water and temperature were also determined, and a monthly environmental productivity index (EPI) was derived that can be used to predict biomass growth. The EPI was calculated as the product of water, temperature, and light indices estimated for conditions at a site in Maricopa (Arizona), and compared with measured biomass at the same site (where the first field trial of A. americana as a crop was completed). The monthly EPI summed over the lifetime of multi-year crops was highly correlated with the average measured biomass of healthy 2- and 3-year-old plants grown in the field. The resulting relationship between EPI and biomass provides a simple model for estimating the production of A. americana at a monthly time step according to light, temperature, and precipitation inputs, and is a useful tool for projecting the potential geographic range of this obligate CAM species in future climatic conditions.

Stomatal response to blue light in crassulacean acid metabolism plants Kalanchoe pinnata and Kalanchoe daigremontiana.

Blue light (BL) is a fundamental cue for stomatal opening in both C3 and C4 plants. However, it is unknown whether crassulacean acid metabolism (CAM) plants open their stomata in response to BL. We investigated stomatal BL responses in the obligate CAM plants Kalanchoe pinnata and Kalanchoe daigremontiana that characteristically open their stomata at night and close them for part of the day, as contrasted with C3 and C4 plants. Stomata opened in response to weak BL superimposed on background red light in both intact leaves and detached epidermal peels of K. pinnata and K. daigremontiana. BL-dependent stomatal opening was completely inhibited by tautomycin and vanadate, which repress type 1 protein phosphatase and plasma membrane H+-ATPase, respectively. The plasma membrane H+-ATPase activator fusicoccin induced stomatal opening in the dark. Both BL and fusicoccin induced phosphorylation of the guard cell plasma membrane H+-ATPase in K. pinnata. These results indicate that BL-dependent stomatal opening occurs in the obligate CAM plants K. pinnata and K. daigremontiana independently of photosynthetic CO2 assimilation mode.

Temporal and spatial transcriptomic and microRNA dynamics of CAM photosynthesis in pineapple.

The altered carbon assimilation pathway of crassulacean acid metabolism (CAM) photosynthesis results in an up to 80% higher water-use efficiency than C3 photosynthesis in plants making it a potentially useful pathway for engineering crop plants with improved drought tolerance. Here we surveyed detailed temporal (diel time course) and spatial (across a leaf gradient) gene and microRNA (miRNA) expression patterns in the obligate CAM plant pineapple [Ananas comosus (L.) Merr.]. The high-resolution transcriptome atlas allowed us to distinguish between CAM-related and non-CAM gene copies. A differential gene co-expression network across green and white leaf diel datasets identified genes with circadian oscillation, CAM-related functions, and source-sink relations. Gene co-expression clusters containing CAM pathway genes are enriched with clock-associated cis-elements, suggesting circadian regulation of CAM. About 20% of pineapple microRNAs have diel expression patterns, with several that target key CAM-related genes. Expression and physiology data provide a model for CAM-specific carbohydrate flux and long-distance hexose transport. Together these resources provide a list of candidate genes for targeted engineering of CAM into C3 photosynthesis crop species.

Adaptation of the obligate CAM plant Clusia alata to light stress: Metabolic responses

In the Crassulacean acid metabolism (CAM) plants Clusia alata Triana and Planch., decarboxylation of citrate during phase III of CAM took place later than malate decarboxylation. The interdependence of these two CO(2) and NADPH sources is discussed. High light accelerated malate decarboxylation during the day and lowered citrate levels. Strong light stress also activated mechanisms that can protect the plant against oxidative stress. Upon transfer from low light (200micromol m(-2)s(-1)) to high light (650-740micromol m(-2)s(-1)), after 2 days, there was a transient increase of non-photochemical quenching (NPQ) of fluorescence of chlorophyll a of photosystem II. This indicated acute photoinhibition, which declined again after 7 days of exposure. Conversely, after 1 week exposure to high light, the mechanisms of interconversion of violaxanthin (V), antheraxanthin (A), zeaxanthin (Z) (epoxydation/de-epoxydation) were activated. This was accompanied by an increase in pigment levels at dawn and dusk.

Impact of developmental stage on CAM expression and growth in an Aechmea hybrid under greenhouse conditions

SummaryBesides environmental factors, expression of crassulacean acid metabolism (CAM) and the related production of malic acid may depend on plant developmental stage. This topic has not yet been investigated under commercial greenhouse conditions. In CAM plant cultivation, malic acid is a central determinant of plant growth, but can also cause physiological leaf damage problems. Here, we present data on diurnal leaf malic acid contents, relative water content dynamics, and measurements of growth at four stages of the plant growth cycle (i.e., ex vitro-acclimated plantlets, 6-month, 12-month, and 18-month-old plants) of Aechmea ‘Maya’. The results obtained showed that Aechmea ‘Maya’ was an obligate CAM plant at all developmental stages during cultivation. Nevertheless, there was some developmental control on the expression of CAM. Under the same environmental conditions, 6-month-old plants accumulated significantly higher amounts of malic acid in their leaves, thus supporting higher growth rates, than in the other growth stages. This larger malic acid pool implied a higher risk of physiological leaf damage, but since remobilisation of malic acid in the early morning remained unaffected during growth, physiological leaf damage might occur throughout vegetative growth.

Cool-night temperature induces spike emergence and affects photosynthetic efficiency and metabolizable carbohydrate and organic acid pools in Phalaenopsis aphrodite

After being acclimated to constant warm (28 degrees C day/28 degrees C night) and cool-night temperature (28 degrees C day/20 degrees C night) regimes in growth chambers for 2 weeks, the two groups of mature Phalaenopsis aphrodite subsp. formosana plants both clearly exhibited a diurnal oscillation of stomatal conductance, net CO(2) uptake rate, malate and starch levels, and the phosphoenolpyruvate carboxylase (EC 4.1.1.31) and NAD(+)-malic enzyme (EC 1.1.1.39) activities. Hence, P. aphrodite is an obligate crassulacean acid metabolism plant. Nevertheless, different night temperature greatly affected both the stomatal conductance and the contribution of ambient and respiratory CO(2) to the nocturnal accumulation of malate. However, the amounts of nocturnal accumulated malate and daily deposited starch appeared to have no significant difference between the two groups. These results demonstrate that P. ahrodite is congruent with the characteristics of CAM plants having great flexibility and plasticity in response to changes in environmental conditions. In addition, the formation of reproductive stem, viz. spike, was noticeably inhibited by a constant warm temperature, but induced by a fluctuating warm day and cool night condition. The relationship between the metabolic pool variation and spike induction of Phalaenopsis is also discussed.

The Effect of Elevated Concentrations of Fructose 2,6-Bisphosphate on Carbon Metabolism during Deacidification in the Crassulacean Acid Metabolism Plant Kalanchöe daigremontiana.

In C(3) plants, the metabolite fructose 2,6-bisphosphate (Fru 2,6-P(2)) has an important role in the regulation of carbon partitioning during photosynthesis. To investigate the impact of Fru 2,6-P(2) on carbon metabolism during Crassulacean acid metabolism (CAM), we have developed an Agrobacterium tumefaciens-mediated transformation system in order to alter genetically the obligate CAM plant Kalanchöe daigremontiana. To our knowledge, this is the first report to use genetic manipulation of a CAM species to increase our understanding of this important form of plant metabolism. Transgenic plants were generated containing a modified rat liver 6-phosphofructo-2-kinase gene. In the plants analyzed the activity of 6-phosphofructo-2-kinase ranged from 175% to 198% of that observed in wild-type plants, resulting in Fru 2,6-P(2) concentrations that were 228% to 350% of wild-type plants after 2 h of illumination. A range of metabolic measurements were made on these transgenic plants to investigate the possible roles of Fru 2,6-P(2) during Suc, starch, and malic acid metabolism across the deacidification period of CAM. The results suggest that Fru 2,6-P(2) plays a major role in regulating partitioning between Suc and starch synthesis during photosynthesis. However, alterations in Fru 2,6-P(2) levels had little effect on malate mobilization during CAM fluxes.

Expression of Modes of Photosynthesis (C3, CAM) inClusia criuvaCamb. in a Cerrado/Gallery Forest Transect

Abstract: The large majority of the Ca. 150 species of the neotropical shrub and tree genus Clusia have the potential to perform Crassulacean acid metabolism (CAM). They are either obligate CAM plants or C3/CAM intermediate plants. Only a very small percentage of the plants studied so far are apparently obligate C3 species. Among these was C. criuva, until recent laboratory studies showed that it may also have a certain CAM capacity under artificial stress conditions. Measurements of stomatal conductance (porometry) and chlorophyll fluorescence variables of C. criuva occurring along a transect from deep shade inside a gallery forest across the semi-shaded ecotone towards a cerrado and into the exposed cerrado itself in central Brazil now show that it can adapt its photosynthetic apparatus to effective performance of C3 photosynthesis under highly different photosynthetic photon flux densities. In addition, however, it does have a certain potential for CAM and cannot be considered as a strictly obligate C3 plant. Should a basic capacity for performing CAM be a general property of the genus, the quest for CAM traits in other remaining putatively obligate C3 species of the genus ought to be pursued.

Expression of Modes of Photosynthesis (C3, CAM) in Clusia criuva Camb. in a CerradolGallery Forest Transect

Abstract: The large majority of the Ca. 150 species of the neotropical shrub and tree genus Clusia have the potential to perform Crassulacean acid metabolism (CAM). They are either obligate CAM plants or C3/CAM intermediate plants. Only a very small percentage of the plants studied so far are apparently obligate C3 species. Among these was C. criuva, until recent laboratory studies showed that it may also have a certain CAM capacity under artificial stress conditions. Measurements of stomatal conductance (porometry) and chlorophyll fluorescence variables of C. criuva occurring along a transect from deep shade inside a gallery forest across the semi‐shaded ecotone towards a cerrado and into the exposed cerrado itself in central Brazil now show that it can adapt its photosynthetic apparatus to effective performance of C3 photosynthesis under highly different photosynthetic photon flux densities. In addition, however, it does have a certain potential for CAM and cannot be considered as a strictly obligate C3 plant. Should a basic capacity for performing CAM be a general property of the genus, the quest for CAM traits in other remaining putatively obligate C3 species of the genus ought to be pursued.

Identification of phosphoenolpyruvate carboxylase isoforms in leaf, stem and roots of the obligate CAM plant Vanilla planifolia Salib. (Orchidaceae): a physiological and molecular approach.

This study provides the first comparative analysis of phosphoenolpyruvate carboxylase isoforms (PEPc; EC 4.1.1.31) in an obligate crassulacean acid metabolism (CAM) plant, Vanilla planifolia Salisb. (Orchidaceae). Nocturnal CO2 fixation and malate accumulation by the leaves and the green stem show that these organs perform CAM. The chloroplast-containing aerial roots, however, exhibit C3 photosynthesis. The catalytic activity of PEPc was highest in the leaves compared with the stem and aerial roots. The Km (PEP) and Ki (malate) were similar in the PEPc extracted from leaf and aerial roots, and significant higher in stem. cDNA was obtained from those tissues and also from the soil-grown roots, and various cDNA clones were detected and amplified by means of RT-PCR and RACE-PCR. The amino-acid sequences of the PEPc isoforms deduced from the cDNA showed a great degree of homology, and Southern blot analysis suggests that the encoding genes form a small multigene family of at least two members. One PEPc isoform (PpcV1) is assumed to be related to CAM because, as shown by northern blot analysis, it is mainly expressed in the CAM-performing organs, i.e. in the leaves and the stem. A further isoform (PpcV2) was identified in the soil-grown roots and aerial roots, but northern blots show that to some extent PpcV2 is also expressed in the leaf and the stem tissues. Thus, it is assumed that PpcV2 encodes the housekeeping isoform of PEPc. Altogether, the present study provides support in favour of the view that isoforms of PEPc are related to specific functions.

Ecophysiology and distribution of the endemic leafless spurgeEuphorbia aphylla and the introducedE. tirucalli (Euphorbiaceae, Euphorbia sect.Tirucalli) in the Canary Islands

The leafless spurgeEuphorbia aphylla (Euphorbiaceae), an endemic species restricted to several of the Canary Islands where it inhabits coastal and arid localities, expresses Crassulacean Acid Metabolism (CAM) when it is subjected to summer drought. A flexible CAM is consistent with the general ecology of the species. It is the only member of sect.Tirucalli native to the Canary Islands and is at the north-western edge of the section's biogeographical range. The other members of the section have a paleotropic distribution and are found throughout Africa. Many of them are regarded as obligate CAM plants, includingE. tirucalli which was used as a comparison in ecophysiological experiments examining the response ofE. aphylla to drought and temperature.

Short-Term Regulation of Crassulacean Acid Metabolism Activity in a Tropical Hemiepiphyte, Clusia uvitana

Diel courses of net CO2 exchange of leaves were studied in Clusia uvitana (Clusiaceae), a tropical Crassulacean acid metabolism (CAM) hemiepiphyte, growing in the crown of a 47-m tall kapok tree on Barro Colorado Island, Panama. Measurements on days without precipitation showed that net uptake of atmospheric CO2 occurred at night, a feature of CAM, as well as in the early morning and late afternoon. During 36 h of almost continuous rainfall, nocturnal net CO2 uptake was abolished and the diel pattern of net CO2 exchange became similar to that of a C3 plant. Exposing well-watered, potted plants of Clusia in the laboratory to temperatures and photosynthetic photon flux densities similar to those during the tropical rainstorm also abolished nocturnal net CO2 uptake. In contrast, Kalanchoe pinnata (Crassulaceae), an obligate CAM plant, still showed net CO2 dark fixation following the same low-light and moderate-temperature conditions, albeit at decreased rates. During these 12-h photoperiods, titratable acidity in Clusia increased slightly above its high level measured at the end of the previous dark period, whereas in Kalanchoe, the acid content decreased by about 40%. A survey among outer canopy leaves of Clusia on Barro Colorado Island showed that leaves that exhibited little or no nocturnal acidification maintained high levels of H+ at dawn and dusk. Progressively lower levels of H+ at dusk were accompanied by progressively higher nocturnal increases in H+. The data suggest that in C. uvitana the rapid switching between CAM- and C3-type carbon fixation that may occur within 24 h in response to environmental changes is controlled by the acidity status of the leaves in the light. Nocturnal CO2 fixation is enhanced by conditions that decrease the organic acid content during the light period.