Constitutive Crassulacean Acid Metabolism Plant Research Articles

The starch-deficient plastidic PHOSPHOGLUCOMUTASE mutant of the constitutive crassulacean acid metabolism (CAM) species Kalanchoë fedtschenkoi impacts diel regulation and timing of stomatal CO2 responsiveness.

Crassulacean acid metabolism (CAM) is a specialized type of photosynthesis characterized by a diel pattern of stomatal opening at night and closure during the day, which increases water-use efficiency. Starch degradation is a key regulator of CAM, providing phosphoenolpyruvate as a substrate in the mesophyll for nocturnal assimilation of CO2. Growing recognition of a key role for starch degradation in C3 photosynthesis guard cells for mediating daytime stomatal opening presents the possibility that starch degradation might also impact CAM by regulating the provision of energy and osmolytes to increase guard cell turgor and drive stomatal opening at night. In this study, we tested the hypothesis that the timing of diel starch turnover in CAM guard cells has been reprogrammed during evolution to enable nocturnal stomatal opening and daytime closure. Biochemical and genetic characterization of wild-type and starch-deficient RNAi lines of Kalanchoë fedtschenkoi with reduced activity of plastidic phosphoglucomutase (PGM) constituted a preliminary approach for the understanding of starch metabolism and its implications for stomatal regulation in CAM plants. Starch deficiency reduced nocturnal net CO2 uptake but had negligible impact on nocturnal stomatal opening. In contrast, daytime stomatal closure was reduced in magnitude and duration in the starch-deficient rPGM RNAi lines, and their stomata were unable to remain closed in response to elevated concentrations of atmospheric CO2 administered during the day. Curtailed daytime stomatal closure was linked to higher soluble sugar contents in the epidermis and mesophyll. Nocturnal stomatal opening is not reliant upon starch degradation, but starch biosynthesis is an important sink for carbohydrates, ensuring daytime stomatal closure in this CAM species.

Time of day and network reprogramming during drought induced CAM photosynthesis in Sedum album.

Plants with facultative crassulacean acid metabolism (CAM) maximize performance through utilizing C3 or C4 photosynthesis under ideal conditions while temporally switching to CAM under water stress (drought). While genome-scale analyses of constitutive CAM plants suggest that time of day networks are shifted, or phased to the evening compared to C3, little is known for how the shift from C3 to CAM networks is modulated in drought induced CAM. Here we generate a draft genome for the drought-induced CAM-cycling species Sedum album. Through parallel sampling in well-watered (C3) and drought (CAM) conditions, we uncover a massive rewiring of time of day expression and a CAM and stress-specific network. The core circadian genes are expanded in S. album and under CAM induction, core clock genes either change phase or amplitude. While the core clock cis-elements are conserved in S. album, we uncover a set of novel CAM and stress specific cis-elements consistent with our finding of rewired co-expression networks. We identified shared elements between constitutive CAM and CAM-cycling species and expression patterns unique to CAM-cycling S. album. Together these results demonstrate that drought induced CAM-cycling photosynthesis evolved through the mobilization of a stress-specific, time of day network, and not solely the phasing of existing C3 networks. These results will inform efforts to engineer water use efficiency into crop plants for growth on marginal land.

Facultative crassulacean acid metabolism (CAM) plants: powerful tools for unravelling the functional elements of CAM photosynthesis

Facultative crassulacean acid metabolism (CAM) describes the optional use of CAM photosynthesis, typically under conditions of drought stress, in plants that otherwise employ C3 or C4 photosynthesis. In its cleanest form, the upregulation of CAM is fully reversible upon removal of stress. Reversibility distinguishes facultative CAM from ontogenetically programmed unidirectional C3-to-CAM shifts inherent in constitutive CAM plants. Using mainly measurements of 24h CO2 exchange, defining features of facultative CAM are highlighted in five terrestrial species, Clusia pratensis, Calandrinia polyandra, Mesembryanthemum crystallinum, Portulaca oleracea and Talinum triangulare. For these, we provide detailed chronologies of the shifts between photosynthetic modes and comment on their usefulness as experimental systems. Photosynthetic flexibility is also reviewed in an aquatic CAM plant, Isoetes howellii. Through comparisons of C3 and CAM states in facultative CAM species, many fundamental biochemical principles of the CAM pathway have been uncovered. Facultative CAM species will be of even greater relevance now that new sequencing technologies facilitate the mapping of genomes and tracking of the expression patterns of multiple genes. These technologies and facultative CAM systems, when joined, are expected to contribute in a major way towards our goal of understanding the essence of CAM.

Limited photosynthetic plasticity in the leaf-succulent CAM plant Agave angustifolia grown at different temperatures.

In Agave angustifolia Haw., a leaf-succulent constitutive crassulacean acid metabolism (CAM) plant of tropical Panama, we tested whether nocturnal CO2 uptake and growth were reduced at night temperatures above 20°C. Unlike some CAM model species from habitats with pronounced day-night temperature variations, in A. angustifolia temperature affected little the relative contributions of CAM and C3 photosynthesis to growth. In plants grown under 12h light/dark regimes of 25/17, 30/22 and 35/27°C, biomass increased with temperature. Maintaining day temperature at 35°C and reducing night temperature from 27 to 17°C markedly lowered growth, a reduction partially reversed when roots were heated to 27°C. Across all treatments, whole-shoot δ13C values ranged between -14.6 and -13.2 ‰, indicating a stable proportion of CO2 was fixed at night, between 75 and 83%. Nocturnal acidification reflected growth, varying between 339 and 393μmol H+ g-1 fresh mass and 63-87μmol H+ cm-2. In outdoor open-top chambers, warming the air 3°C above ambient at night did not reduce biomass accumulation. The persistence of a high capacity for nocturnal CO2 fixation at the expense of a limited capacity for switching between C3 and CAM probably makes this Agave, and others like it, potential species for biomass production in seasonally-dry landscapes.

Crassulacean acid metabolism-cycling in Euphorbia milii.

Crassulacean acid metabolism (CAM) occurs in many Euphorbiaceae, particularly Euphorbia, a genus with C3 and C4 species as well. With the aim of contributing to our knowledge of the evolution of CAM in this genus, this study examined the possible occurrence of CAM in Euphorbia milii, a species with leaf succulence and drought tolerance suggestive of this carbon fixation pathway. Leaf anatomy consisted of a palisade parenchyma, a spongy parenchyma and a bundle sheath with chloroplasts, which indicates the possible functioning of C2 photosynthesis. No evidence of nocturnal CO2 fixation was found in plants of E. milii either watered or under drought; watered plants had a low nocturnal respiration rate (R). After 12 days without watering, the photosynthetic rate (P N) decreased 85 % and nocturnal R was nearly zero. Nocturnal H(+) accumulation (ΔH(+)) in watered plants was 18 ± 2 (corresponding to malate) and 18 ± 4 (citrate) μmol H(+) (g fresh mass)(-1). Respiratory CO2 recycling through acid synthesis contributed to a night-time water saving of 2 and 86 % in watered plants and plants under drought, respectively. Carbon isotopic composition (δ(13)C) was -25.2 ± 0.7 ‰ in leaves and -24.7 ± 0.1 ‰ in stems. Evidence was found for the operation of weak CAM in E. milii, with statistically significant ΔH(+), no nocturnal CO2 uptake and values of δ(13)C intermediate between C3 and constitutive CAM plants; ΔH(+) was apparently attributable to both malate and citrate. The results suggest that daily malate accumulation results from recycling of part of the nocturnal respiratory CO2, which helps explain the occurrence of an intermediate value of leaf δ(13)C. Euphorbia milii can be considered as a CAM-cycling species. The significance of the operation of CAM-cycling in E. milii lies in water conservation, rather than carbon acquisition. The possible occurrence of C2 photosynthesis merits research.

Seasonal photosynthetic gas exchange and water-use efficiency in a constitutive CAM plant, the giant saguaro cactus (Carnegiea gigantea)

Crassulacean acid metabolism (CAM) and the capacity to store large quantities of water are thought to confer high water use efficiency (WUE) and survival of succulent plants in warm desert environments. Yet the highly variable precipitation, temperature and humidity conditions in these environments likely have unique impacts on underlying processes regulating photosynthetic gas exchange and WUE, limiting our ability to predict growth and survival responses of desert CAM plants to climate change. We monitored net CO(2) assimilation (A(net)), stomatal conductance (g(s)), and transpiration (E) rates periodically over 2 years in a natural population of the giant columnar cactus Carnegiea gigantea (saguaro) near Tucson, Arizona USA to investigate environmental and physiological controls over carbon gain and water loss in this ecologically important plant. We hypothesized that seasonal changes in daily integrated water use efficiency (WUE(day)) in this constitutive CAM species would be driven largely by stomatal regulation of nighttime transpiration and CO(2) uptake responding to shifts in nighttime air temperature and humidity. The lowest WUE(day) occurred during time periods with extreme high and low air vapor pressure deficit (D(a)). The diurnal with the highest D(a) had low WUE(day) due to minimal net carbon gain across the 24 h period. Low WUE(day) was also observed under conditions of low D(a); however, it was due to significant transpiration losses. Gas exchange measurements on potted saguaro plants exposed to experimental changes in D(a) confirmed the relationship between D(a) and g(s). Our results suggest that climatic changes involving shifts in air temperature and humidity will have large impacts on the water and carbon economy of the giant saguaro and potentially other succulent CAM plants of warm desert environments.

Conserved and Divergent Rhythms of Crassulacean Acid Metabolism-Related and Core Clock Gene Expression in the Cactus Opuntia ficus-indica

The cactus Opuntia ficus-indica is a constitutive Crassulacean acid metabolism (CAM) species. Current knowledge of CAM metabolism suggests that the enzyme phosphoenolpyruvate carboxylase kinase (PPCK) is circadian regulated at the transcriptional level, whereas phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH), NADP-malic enzyme (NADP-ME), and pyruvate phosphate dikinase (PPDK) are posttranslationally controlled. As little transcriptomic data are available from obligate CAM plants, we created an expressed sequence tag database derived from different organs and developmental stages. Sequences were assembled, compared with sequences in the National Center for Biotechnology Information nonredundant database for identification of putative orthologs, and mapped using Kyoto Encyclopedia of Genes and Genomes Orthology and Gene Ontology. We identified genes involved in circadian regulation and CAM metabolism for transcriptomic analysis in plants grown in long days. We identified stable reference genes for quantitative polymerase chain reaction and found that OfiSAND, like its counterpart in Arabidopsis (Arabidopsis thaliana), and OfiTUB are generally appropriate standards for use in the quantification of gene expression in O. ficus-indica. Three kinds of expression profiles were found: transcripts of OfiPPCK oscillated with a 24-h periodicity; transcripts of the light-active OfiNADP-ME and OfiPPDK genes adapted to 12-h cycles, while transcript accumulation patterns of OfiPEPC and OfiMDH were arrhythmic. Expression of the circadian clock gene OfiTOC1, similar to Arabidopsis, oscillated with a 24-h periodicity, peaking at night. Expression of OfiCCA1 and OfiPRR9, unlike in Arabidopsis, adapted best to a 12-h rhythm, suggesting that circadian clock gene interactions differ from those of Arabidopsis. Our results indicate that the evolution of CAM metabolism could be the result of modified circadian regulation at both the transcriptional and posttranscriptional levels.

Two phosphoenolpyruvate carboxykinases coexist in the Crassulacean Acid Metabolism plant Ananas comosus. Isolation and characterization of the smaller 65kDa form

Two phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.49) isoforms of 74 and 65 kDa were found to coexist in vivo in pineapple leaves, a constitutive Crassulacean Acid Metabolism plant. The 65 kDa form was not the result of proteolytic cleavage of the larger form since extraction methods reported to prevent PEPCK proteolysis in other plant tissues failed to yield a single immunoreactive PEPCK polypeptide in leaf extracts. In this work, the smaller form of 65 kDa was purified to homogeneity and physically and kinetically characterized and showed parameters compatible with a fully active enzyme. The specific activity was nearly twice higher for decarboxylation of oxaloacetate when compared to carboxylation of phosphoenolpyruvate. Kinetic parameters fell within the range of those estimated for other plant PEPCKs. Its activity was affected by several metabolites, as shown by inhibition by 3-phosphoglycerate, citrate, malate, fructose-1,6-bisphosphate, l-asparagine and activation of the decarboxylating activity by succinate. A break in the Arrhenius plot at about 30°C indicates that PEPCK structure is responsive to changes in temperature. The results indicate that pineapple leaves contain two PEPCK forms. The biochemical characterization of the smaller isoform performed in this work suggests that it could participate in both carbon and nitrogen metabolism in vivo by acting as a decarboxylase.

Research note: Large gene family of phosphoenolpyruvate carboxylase in the crassulacean acid metabolism plant Kalanchoe pinnata (Crassulaceae) characterised by partial cDNA sequence analysis.

Clones coding for a 1100-bp cDNA sequence of phosphoenolpyruvate carboxylase (PEPC) of the constitutive crassulacean acid metabolism (CAM) plant Kalanchoe pinnata (Lam.) Pers., were isolated by reverse transcription-polymerase chain reaction (RT-PCR) and characterised by restriction fragment length polymorphism analysis and DNA sequencing. Seven distinct PEPC isogenes were recovered, four in leaves and three in roots (EMBL accession numbers: AJ344052-AJ344058). Sequence similarity comparisons and distance neighbour-joining calculations separate the seven PEPC isoforms into two clades, one of which contains the three PEPCs found in roots. The second clade contains the four isoforms found in leaves and is divided into two branches, one of which contains two PEPCs most similar with described previously CAM isoforms. Of these two isoforms, however, only one exhibited abundant expression in CAM-performing leaves, but not in very young leaves, which do not exhibit CAM, suggesting this isoform encodes a CAM-specific PEPC. Protein sequence calculations suggest that all isogenes are likely derived from a common ancestor gene, presumably by serial gene duplication events. To our knowledge, this is the most comprehensive identification of a PEPC gene family from a CAM plant, and the greatest number of PEPC isogenes reported for any vascular plant to date.

Regulatory protein phosphorylation of phosphoenolpyruvate carboxylase in the facultative crassulacean-acid-metabolism plant Mesembryanthemum crystallinum L.

Phosphoenolpyruvate PyrP carboxylase (PyrPC) and PyrPC kinase were copurified from dark-adapted leaves of the common ice plant Mesembryanthemum crystallinum L. with crassulacean-acid metabolism (CAM). Purification by (NH4)2SO4 fractionation, chromatography on Fractogel-DEAE and hydroxylapatite resulted in a PyrPC preparation with a specific activity of 23-25 U/mg protein and a protein kinase activity of 255 mumol Pi.mol-1 PyrPC.s-1. After in vitro phosphorylation, the most prominently phosphorylated polypeptide was identified as PyrPC by immunoblotting and sequencing. Phosphorylation of PyrPC in vitro by incubation with 400 microM MgATP decreased its sensitivity towards malate. When purified in the absence of the protease inhibitor chymostatin, PyrPC lost an N-terminal sequence of 128 amino acids. Although the carboxylation reaction was unaffected, the truncated PyrPC could neither be phosphorylated in vitro nor inhibited by malate. This result and data obtained by limited proteolysis concur with the hypothesis [Jiao, J.A. & Chollet, R. (1989) Arch. Biochem. Biophys. 283, 300-305] that Ser11 is the phosphorylation site of the CAM PyrPC of M. crystallinum. At pH 7.0, the Km for ATP of the protein kinase was 25 microM; phosphorylation of PyrPC was maximal after 30 min at pH 7.0. The kinase showed also activity with histone III-S but not with dephosphorylated casein. It was inhibited by malate. The results show, that reversible protein phosphorylation is an important factor in the regulation of PyrPC in the facultative CAM plant M. crystallinum, similar to C4 and constitutive CAM plants.

Activity and quantity of ribulose bisphosphate carboxylase-and phosphoenolpyruvate carboxylase-protein in two Crassulacean acid metabolism plants in relation to leaf age, nitrogen nutrition, and point in time during a day/night cycle

Activity of ribulose 1,5-bisphosphate (RuBP) carboxylase in leaf extracts of the constitutive Crassulacean acid metabolism (CAM) plant Kalanchoe pinnata (Lam.) Pers. decreased with increasing leaf age, whereas the activity of phosphoenolpyruvate (PEP) carboxylase increased. Changes in enzyme activities were associated with changes in the amount of enzyme proteins as determined by immunochemical analysis, sucrose density gradient centrifugation, and SDS gel electrophoresis of leaf extracts. Young developing leaves of plants which received high amounts of NO 3 (-) during growth contained about 30% of the total soluble protein in the form of RuBP carboxylase; this value declined to about 17% in mature leaves. The level of PEP carboxylase in young leaves of plants at high NO 3 (-) was an estimated 1% of the total soluble protein and increased to approximately 10% in mature leaves, which showed maximum capacity for dark CO2 fixation. The growth of plants at low levels of NO 3 (-) decreased the content of soluble protein per unit leaf area as well as the extractable activity and the percentage contribution of both RUBP carboxylase and PEP carboxylase to total soluble leaf protein. There was no definite change in the ratio of RuBP carboxylase to PEP carboxylase activity with a varying supply of NO 3 (-) during growth. It has been suggested (e.g., Planta 144, 143-151, 1978) that a rhythmic pattern of synthesis and degradation of PEP carboxylase protein is involved in the regulation of β-carboxylation during a day/night cycle in CAM. No such changes in the quantity of PEP carboxylase protein were observed in the leaves of Kalanchoe pinnata (Lam.) Pers. or in the leaves of the inducible CAM plant Mesembryanthemum crystallinum L.