[Show abstract][Hide abstract] ABSTRACT: Phosphoenolpyruvate carboxylase (PEPC) catalyses the initial fixation of atmospheric CO2 into oxaloacetate and subsequently malate. Nocturnal accumulation of malic acid within the vacuole of photosynthetic cells is a typical feature of plants that perform crassulacean acid metabolism (CAM). PEPC is a ubiquitous plant enzyme encoded by a small gene family, and each member encodes an isoform with specialized function. CAM-specific PEPC isoforms probably evolved from ancestral non-photosynthetic isoforms by gene duplication events and subsequent acquisition of transcriptional control elements that mediate increased leaf-specific or photosynthetic-tissue-specific mRNA expression. To understand the patterns of functional diversification related to the expression of CAM, ppc gene families and photosynthetic patterns were characterized in 11 closely related orchid species from the subtribe Oncidiinae with a range of photosynthetic pathways from C3 photosynthesis (Oncidium cheirophorum, Oncidium maduroi, Rossioglossum krameri, and Oncidium sotoanum) to weak CAM (Oncidium panamense, Oncidium sphacelatum, Gomesa flexuosa and Rossioglossum insleayi) and strong CAM (Rossioglossum ampliatum, Trichocentrum nanum, and Trichocentrum carthagenense). Phylogenetic analysis revealed the existence of two main ppc lineages in flowering plants, two main ppc lineages within the eudicots, and three ppc lineages within the Orchidaceae. Our results indicate that ppc gene family expansion within the Orchidaceae is likely to be the result of gene duplication events followed by adaptive sequence divergence. CAM-associated PEPC isoforms in the Orchidaceae probably evolved from several independent origins.
Journal of Experimental Botany 06/2014; · 5.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To meet future food and energy security needs, which are amplified by increasing population growth and reduced natural resource availability, metabolic engineering efforts have moved from manipulating single genes/proteins to introducing multiple genes and novel pathways to improve photosynthetic efficiency in a more comprehensive manner. Biochemical carbon-concentrating mechanisms such as crassulacean acid metabolism (CAM), which improves photosynthetic, water-use, and possibly nutrient-use efficiency, represent a strategic target for synthetic biology to engineer more productive C3 crops for a warmer and drier world. One key challenge for introducing multigene traits like CAM onto a background of C3 photosynthesis is to gain a better understanding of the dynamic spatial and temporal regulatory events that underpin photosynthetic metabolism. With the aid of systems and computational biology, vast amounts of experimental data encompassing transcriptomics, proteomics, and metabolomics can be related in a network to create dynamic models. Such models can undergo simulations to discover key regulatory elements in metabolism and suggest strategic substitution or augmentation by synthetic components to improve photosynthetic performance and water-use efficiency in C3 crops. Another key challenge in the application of synthetic biology to photosynthesis research is to develop efficient systems for multigene assembly and stacking. Here, we review recent progress in computational modelling as applied to plant photosynthesis, with attention to the requirements for CAM, and recent advances in synthetic biology tool development. Lastly, we discuss possible options for multigene pathway construction in plants with an emphasis on CAM-into-C3 engineering.
Journal of Experimental Botany 02/2014; · 5.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Climatic extremes threaten agricultural sustainability worldwide. One approach to increase plant water- use efficiency (WUE) is to introduce crassulacean acid metabolism (CAM) into C3 crops. Such a task requires comprehensive systems-level understanding of the enzymatic and regulatory pathways underpinning this temporal CO2 pump. Here we review the progress that has been made in achieving this goal. Given that CAM arose through multiple independent evolutionary ori- gins, comparative transcriptomics and genomics of taxonomically diverse CAM species are being used to define the genetic ‘parts list’ required to operate the core CAM functional modules of nocturnal carboxylation, diurnal decarboxylation, and inverse stomatal regulation. Engi- neered CAM offers the potential to sustain plant productivity for food, feed, fiber, and biofuel production in hotter and drier climates.
Trends in Plant Science 02/2014; 19(6). · 11.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Over 13% of all genes in the Arabidopsis thaliana genome encode for proteins classified as having a completely unknown function, with the function of >30% of the Arabidopsis proteome poorly characterized. Although empirical data in the form of mRNA and proteome profiling experiments suggest that many of these proteins play an important role in different biological processes, their functional characterization remains one of the major challenges in modern biology. To expand the annotation of genes with unknown function involved in the response of Arabidopsis to different environmental stress conditions, we selected 1007 such genes and tested the response of their corresponding homozygous T-DNA insertional mutants to salinity, oxidative, osmotic, heat, cold and hypoxia stresses. Depending on the specific abiotic stresses tested, 12-31% of mutants had an altered stress-response phenotype. Interestingly, 832 out of 1007 mutants showed tolerance or sensitivity to more than one abiotic stress treatment, suggesting that genes of unknown function could play an important role in abiotic stress-response signaling, or general acclimation mechanisms. Further analysis of multiple stress-response phenotypes within different populations of mutants revealed interesting links between acclimation to heat, cold and oxidative stresses, as well as between sensitivity to ABA, osmotic, salinity, oxidative and hypoxia stresses. Our findings provide a significant contribution to the biological characterization of genes with unknown function in Arabidopsis and demonstrate that many of these genes play a key role in the response of plants to abiotic stresses.
Physiologia Plantarum 03/2013; · 3.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aerobic metabolism of vertebrates is linked to membrane fatty acid (FA) composition. Although the membrane pacemaker hypothesis posits that desaturation of FAs accounts for variation in resting or basal metabolic rate (BMR), little is known about the FA profiles that underpin variation in maximal metabolic rate (MMR). We examined membrane FA composition of liver and skeletal muscle in mice after seven generations of selection for increased MMR. In both liver and skeletal muscle, unsaturation index did not differ between control and high-MMR mice. We also examined membrane FA composition at the individual-level of variation. In liver, 18:0, 20:3 n-6, 20:4 n-6, and 22:6 n-3 FAs were significant predictors of MMR. In gastrocnemius muscle, 18:2 n-6, 20:4 n-6, and 22:6 n-3 FAs were significant predictors of MMR. In addition, muscle 16:1 n-7, 18:1 n-9, and 22:5 n-3 FAs were significant predictors of BMR, whereas no liver FAs were significant predictors of BMR. Our findings indicate that (i) individual variation in MMR and BMR appears to be linked to membrane FA composition in the skeletal muscle and liver, and (ii) FAs that differ between selected and control lines are involved in pathways that can affect MMR or BMR.
Comparative biochemistry and physiology. Part A, Molecular & integrative physiology 02/2013; · 2.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The development of low water input forages would be useful for improving the
water-use efficiency of livestock production in semi-arid and arid regions. The
desiccation-tolerant (DT) species Sporobolus stapfianus Gandoger and two desic-
cation-sensitive (DS) species, Sporobolus pyramidalis and Sporobolus fimbriatus
(Trin.) Nees. (Poaceae), were evaluated for aerial biomass production and seed
productivity under three different irrigation regimes. Sporobolus stapfianus dis-
played the least biomass production, whereas S. pyramidalis and S. fimbriatus
produced up to 3.8- and 11.2-fold greater dry biomass, respectively, at the highest
irrigation rate of 12 334 l (0.01 acre-feet). Sporobolus fimbriatus and to a lesser
extent S. pyramidalis showed significant increases in biomass production in
response to increased irrigation rates, whereas S. stapfianus did not. Sporobolus
pyramidalis and S. fimbriatus exhibited 3.2- and 6.0-fold greater seed production,
respectively, in response to increased irrigation rates, whereas S. stapfianus
showed only a 1.4-fold increase. All Sporobolus species possessed forage quality
traits (e.g. crude protein, fibre content) comparable to those of timothy, a forage
grass grown widely in the Great Basin in the western United States. Micronutrient
content exceeded the minimum requirements of beef cattle, without surpassing
tolerable limits, with the exception of zinc, which appeared low in all three Sporo-
bolus species. The low water requirements displayed by these species, combined
with their acceptable forage qualities, indicate that these grasses have the poten-
tial to serve farmers and ranchers in semi-arid and arid regions of the western
United States where irrigation resources are limited.
Journal of Agronomy and Crop Science 01/2013; · 2.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Selaginella lepidophylla is one of only a few species of spike mosses (Selaginellaceae) that have evolved desiccation tolerance (DT) or the ability to 'resurrect' from an air-dried state. In order to understand the metabolic basis of DT, S. lepidophylla was subjected to a five-stage, rehydration/dehydration cycle, then analyzed using non-biased, global metabolomics profiling technology based on GC/MS and UHLC/MS/MS2 platforms. A total of 251 metabolites including 167 named (66.5%) and 84 (33.4%) unnamed compounds were characterized. Only 42 (16.7%) and 74 (29.5%) of compounds showed significantly increased or decreased abundance, respectively, indicating that most compounds were produced constitutively including highly abundant trehalose, sucrose, and glucose. Several glycolysis/ gluconeogenesis and tricarboxylic acid (TCA) cycle intermediates showed increased abundance at 100% relative water content (RWC) and 50% RWC. Vanillate, a potent antioxidant was also more abundant in the hydrated state. Many different sugar alcohols and sugar acids were more abundant in the hydrated state. These polyols likely decelerate the rate of water loss during the drying process as well as slow water absorption during rehydration, stabilize proteins, and scavenge reactive oxygen species (ROS). In contrast, nitrogen-rich and γ-glutamyl amino acids, citrulline and nucleotide catabolism products (e.g., allantoin) were more abundant in the dry states, suggesting that these compounds might play important roles in nitrogen remobilization during rehydration or in ROS scavenging. UV-protective compounds such as 3-(3-hydroxyphenyl)propionate, apigenin and naringenin, were more abundant in the dry states. Most lipids were produced constitutively with the exception of choline phosphate, which likely plays a role in membrane hydration and stabilization. In contrast, several polyunsaturated fatty acids were more abundant in the hydrated states, suggesting that these compounds likely help maintain membrane fluidity during dehydration. Lastly, S. lepidophylla contained 7 unnamed compounds that displayed 2-fold or greater abundance in dry or rehydrating states, suggesting that these compounds might play adaptive roles in DT.
[Show abstract][Hide abstract] ABSTRACT: Spike mosses (Selaginellaceae) represent an ancient lineage of vascular plants in which some species have evolved desiccation tolerance (DT). A sister-group contrast to reveal the metabolic basis of DT was conducted between a desiccation-tolerant species, Selaginella lepidophylla, and a desiccation-sensitive species, Selaginella moellendorffii, at 100% relative water content (RWC) and 50% RWC using non-biased, global metabolomics profiling technology, based on GC/MS and UHLC/MS/MS(2) platforms. A total of 301 metabolites, including 170 named (56.5%) and 131 (43.5%) unnamed compounds, were characterized across both species. S. lepidophylla retained significantly higher abundances of sucrose, mono- and polysaccharides, and sugar alcohols than did S. moellendorffii. Aromatic amino acids, the well-known osmoprotectant betaine and flavonoids were also more abundant in S. lepidophylla. Notably, levels of γ-glutamyl amino acid, linked with glutathione metabolism in the detoxification of reactive oxygen species, and with possible nitrogen remobilization following rehydration, were markedly higher in S. lepidophylla. Markers for lipoxygenase activity were also greater in S. lepidophylla, especially at 50% RWC. S. moellendorffii contained more than twice the number of unnamed compounds, with only a slightly greater abundance than in S. lepidophylla. In contrast, S. lepidophylla contained 14 unnamed compounds of fivefold or greater abundance than in S. moellendorffii, suggesting that these compounds might play critical roles in DT. Overall, S. lepidophylla appears poised to tolerate desiccation in a constitutive manner using a wide range of metabolites with some inducible components, whereas S. moellendorffii mounts only limited metabolic responses to dehydration stress.
[Show abstract][Hide abstract] ABSTRACT: Isolation of high quality, high molecular weight RNA from plant tissues is complicated by high levels of polyphenols and polysaccharides
which bind to and/or co-precipitate with RNA. Using high molecular weight polyethylene glycol (HMW-PEG), RNA was successfully
isolated from plant species in which other RNA extraction methods and commercially available kits failed to deliver suitable
results. We tested various buffer systems and isolation conditions with and without PEG or PVP (polyvinylpyrrolidone) using
tissue from species ofAloe, Ananas, Clusia, Euphorbia, Kalanchoe, Opuntia, andPyrrosia, all of which contain high amounts of phenolic compounds and/or polysaccharides. HMW-PEG was found to be more effective than
PVP in removing these compounds. RNA extraction using HMW-PEG resulted in RNA of high quality from all species investigated,
as indicated by UV light absorption profiles, and also yielded PCR amplification products after reverse transcription.
[Show abstract][Hide abstract] ABSTRACT: In the halophytic species Mesembryanthemum crystallinum, the induction of crassulacean acid metabolism (CAM) by salinity requires a substantial investment of resources in storage carbohydrates to provide substrate for nocturnal CO(2) uptake. Acclimation to salinity also requires the synthesis and accumulation of cyclitols as compatible solutes, maintenance of root respiration, and nitrate assimilation. This study assessed the hierarchy and coordination of sinks for carbohydrate in leaves and roots during acclimation to salinity in M. crystallinum. By comparing wild type and a CAM-/starch-deficient mutant of this species, it was sought to determine if other metabolic sinks could compensate for a curtailment in CAM and enable acclimation to salinity. Under salinity, CAM deficiency reduced 24 h photosynthetic carbon gain by >50%. Cyclitols were accumulated to comparable levels in leaves and roots of both the wild type and mutant, but represented only 5% of 24 h carbon balance. Dark respiration of leaves and roots was a stronger sink for carbohydrate in the mutant compared with the wild type and implied higher maintenance costs for the metabolic processes underpinning acclimation to salinity when CAM was curtailed. CAM required the nocturnal mobilization of >70% of primary carbohydrate in the wild type and >85% of carbohydrate in the mutant. The substantial allocation of carbohydrate to CAM limited the export of sugars to roots, and the root:shoot ratio declined under salinity. The data suggest a key role for the vacuole in regulating the supply and demand for carbohydrate over the day/night cycle in the starch-/CAM-deficient mutant.
Journal of Experimental Botany 01/2012; 63(5):1985-96. · 5.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The capture and utilization of light is an exquisitely evolved process. The single-component microbial opsins, although more limited than multicomponent cascades in processing, display unparalleled compactness and speed. Recent advances in understanding microbial opsins have been driven by molecular engineering for optogenetics and by comparative genomics. Here we provide a Primer on these light-activated ion channels and pumps, describe a group of opsins bridging prior categories, and explore the convergence of molecular engineering and genomic discovery for the utilization and understanding of these remarkable molecular machines.
[Show abstract][Hide abstract] ABSTRACT: Chilling and freezing can reduce significantly vine survival and fruit set in Vitis vinifera wine grape. To overcome such production losses, a recently identified grapevine C-repeat binding factor (CBF) gene, VvCBF4, was overexpressed in grape vine cv. 'Freedom' and found to improve freezing survival and reduced freezing-induced electrolyte leakage by up to 2 °C in non-cold-acclimated vines. In addition, overexpression of this transgene caused a reduced growth phenotype similar to that observed for CBF overexpression in Arabidopsis and other species. Both freezing tolerance and reduced growth phenotypes were manifested in a transgene dose-dependent manner. To understand the mechanistic basis of VvCBF4 transgene action, one transgenic line (9-12) was genotyped using microarray-based mRNA expression profiling. Forty-seven and 12 genes were identified in unstressed transgenic shoots with either a >1.5-fold increase or decrease in mRNA abundance, respectively. Comparison of mRNA changes with characterized CBF regulons in woody and herbaceous species revealed partial overlaps, suggesting that CBF-mediated cold acclimation responses are widely conserved. Putative VvCBF4-regulon targets included genes with functions in cell wall structure, lipid metabolism, epicuticular wax formation and stress-responses suggesting that the observed cold tolerance and dwarf phenotypes are the result of a complex network of diverse functional determinants.
[Show abstract][Hide abstract] ABSTRACT: Understanding how plants tolerate dehydration is a prerequisite for developing novel strategies for improving drought tolerance. The desiccation-tolerant (DT) Sporobolus stapfianus and the desiccation-sensitive (DS) Sporobolus pyramidalis formed a sister group contrast to reveal adaptive metabolic responses to dehydration using untargeted global metabolomic analysis. Young leaves from both grasses at full hydration or at 60% relative water content (RWC) and from S. stapfianus at lower RWCs were analyzed using liquid and gas chromatography linked to mass spectrometry or tandem mass spectrometry. Comparison of the two species in the fully hydrated state revealed intrinsic differences between the two metabolomes. S. stapfianus had higher concentrations of osmolytes, lower concentrations of metabolites associated with energy metabolism, and higher concentrations of nitrogen metabolites, suggesting that it is primed metabolically for dehydration stress. Further reduction of the leaf RWC to 60% instigated a metabolic shift in S. stapfianus toward the production of protective compounds, whereas S. pyramidalis responded differently. The metabolomes of S. stapfianus leaves below 40% RWC were strongly directed toward antioxidant production, nitrogen remobilization, ammonia detoxification, and soluble sugar production. Collectively, the metabolic profiles obtained uncovered a cascade of biochemical regulation strategies critical to the survival of S. stapfianus under desiccation.
The Plant Cell 04/2011; 23(4):1231-48. · 9.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The physiology of the unicellular green alga Dunaliella salina in response to abiotic stress has been studied for sev-eral decades. Early D. salina research focused on its remarkable salinity tolerance and ability, upon exposure to vari-ous abiotic stresses, to accumulate high concentrations of β-carotene and other carotenoid pigments valued highly as nutraceuticals. The simple life cycle and growth requirements of D. salina make this organism one of the large-scale commercially exploited microalgae for natural carotenoids. Recent advances in genomics and proteomics now allow investigation of abiotic stress responses at the molecular level. Detailed knowledge of isoprenoid biosynthesis mecha-nisms and the development of molecular tools and techniques for D. salina will allow the improvement of physiological characteristics of algal strains and the use of transgenic algae in bioreactors. Here we review D. salina isoprenoid and carotenoid biosynthesis regulation, and also the biotechnological and genetic transformation procedures developed for this alga that set the stage for its future use as a production system.
[Show abstract][Hide abstract] ABSTRACT: Abiotic stresses, such as water deficit and soil salinity, result in changes in physiology, nutrient use, and vegetative growth in vines, and ultimately, yield and flavor in berries of wine grape, Vitis vinifera L. Large-scale expressed sequence tags (ESTs) were generated, curated, and analyzed to identify major genetic determinants responsible for stress-adaptive responses. Although roots serve as the first site of perception and/or injury for many types of abiotic stress, EST sequencing in root tissues of wine grape exposed to abiotic stresses has been extremely limited to date. To overcome this limitation, large-scale EST sequencing was conducted from root tissues exposed to multiple abiotic stresses.
A total of 62,236 expressed sequence tags (ESTs) were generated from leaf, berry, and root tissues from vines subjected to abiotic stresses and compared with 32,286 ESTs sequenced from 20 public cDNA libraries. Curation to correct annotation errors, clustering and assembly of the berry and leaf ESTs with currently available V. vinifera full-length transcripts and ESTs yielded a total of 13,278 unique sequences, with 2302 singletons and 10,976 mapped to V. vinifera gene models. Of these, 739 transcripts were found to have significant differential expression in stressed leaves and berries including 250 genes not described previously as being abiotic stress responsive. In a second analysis of 16,452 ESTs from a normalized root cDNA library derived from roots exposed to multiple, short-term, abiotic stresses, 135 genes with root-enriched expression patterns were identified on the basis of their relative EST abundance in roots relative to other tissues.
The large-scale analysis of relative EST frequency counts among a diverse collection of 23 different cDNA libraries from leaf, berry, and root tissues of wine grape exposed to a variety of abiotic stress conditions revealed distinct, tissue-specific expression patterns, previously unrecognized stress-induced genes, and many novel genes with root-enriched mRNA expression for improving our understanding of root biology and manipulation of rootstock traits in wine grape. mRNA abundance estimates based on EST library-enriched expression patterns showed only modest correlations between microarray and quantitative, real-time reverse transcription-polymerase chain reaction (qRT-PCR) methods highlighting the need for deep-sequencing expression profiling methods.
[Show abstract][Hide abstract] ABSTRACT: The impact of water deficit on stilbene biosynthesis in wine grape (Vitis vinifera) berries was investigated. Water deficit increased the accumulation of trans-piceid (the glycosylated form of resveratrol) by 5-fold in Cabernet Sauvignon berries but not in Chardonnay. Similarly, water deficit significantly increased the transcript abundance of genes involved in the biosynthesis of stilbene precursors in Cabernet Sauvignon. Increased expression of stilbene synthase, but not that of resveratrol-O-glycosyltransferase, resulted in increased trans-piceid concentrations. In contrast, the transcript abundance of the same genes declined in Chardonnay in response to water deficit. Twelve single nucleotide polymorphisms (SNPs) were identified in the promoters of stilbene synthase genes of Cabernet Sauvignon, Chardonnay, and Pinot Noir. These polymorphisms resulted in eight changes within the predicted cis regulatory elements in Cabernet Sauvignon and Chardonnay. These results suggest that cultivar-specific molecular mechanisms might exist that control resveratrol biosynthesis in grapes.
Journal of Agricultural and Food Chemistry 01/2011; 59(1):289-97. · 2.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The identification of substrates represents a critical challenge for understanding any protein kinase-based signal transduction pathway. In Arabidopsis, there are more than 1000 different protein kinases, 34 of which belong to a family of Ca(2+)-dependent protein kinases (CPKs). While CPKs are implicated in regulating diverse aspects of plant biology, from ion transport to transcription, relatively little is known about isoform-specific differences in substrate specificity, or the number of phosphorylation targets. Here, in vitro kinase assays were used to compare phosphorylation targets of four CPKs from Arabidopsis (CPK1, 10, 16, and 34). Significant differences in substrate specificity for each kinase were revealed by assays using 103 different substrates. For example CPK16 phosphorylated Serine 109 in a peptide from the stress-regulated protein, Di19-2 with K(M) ∼70 μM, but this site was not phosphorylated significantly by CPKs 1, 10, or 34. In contrast, CPKs 1, 10, and 34 phosphorylated 93 other peptide substrates not recognized by CPK16. Examples of substrate specificity differences among all four CPKs were verified by kinetic analyses. To test the correlation between in vivo phosphorylation events and in vitro kinase activities, assays were performed with 274 synthetic peptides that contained phosphorylation sites previously mapped in proteins isolated from plants (in vivo-mapped sites). Of these, 74 (27%) were found to be phosphorylated by at least one of the four CPKs tested. This 27% success rate validates a robust strategy for linking the activities of specific kinases, such as CPKs, to the thousands of in planta phosphorylation sites that are being uncovered by emerging technologies.
Frontiers in Plant Science 01/2011; 2:36. · 3.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Desiccation tolerance (DT) is defined as the equilibration of protoplasmic water potential with that of the surrounding air
(generally dry) without loss of viability upon rehydration. Vegetative DT is widespread among mosses and lichens, but is relatively
rare in vascular plants (0.15%). Recent studies of selected resurrection species indicate that while resurrection plants might
have evolved unique adaptive proteins, enzymes, and antioxidants, the molecular genetic basis of DT lies in the orchestration
of transcriptional and posttranscriptional regulatory programs that operate during drying and rehydration. DT requires signaling
pathways and regulatory mechanisms, aspects of which resemble developmental programs present in orthodox seeds, which result
in the accumulation of oligosaccharides, stress adaptive proteins, antioxidants, reactive oxygen scavenging enzymes, as well
as alterations in the composition and structure of membrane lipids. Functional genomics studies using transcriptome, proteome,
and metabolome analyses are just beginning to unravel the system complexity required to orchestrate the metabolic symphony
that is DT. The status of current gene discovery efforts is summarized along with major transcriptome technologies available
currently to conduct desiccation sensitive versus tolerant species comparisons. These strategies, integrated with large-scale
proteomic and metabolomic investigations currently in progress, promise to revolutionize our understanding of the mechanistic
basis of desiccation tolerance in resurrection plants.
[Show abstract][Hide abstract] ABSTRACT: Crassulacean acid metabolism (CAM) is a specialised mode of photosynthesis that improves atmospheric CO 2 assimilation in water-limited terrestrial and epiphytic habitats and in CO 2 -limited aquatic environments. In contrast with C 3 and C 4 plants, CAM plants take up CO 2 from the atmosphere partially or predominantly at night. CAM is taxonomically widespread among vascular plants and is present in many succulent species that occupy semiarid regions, as well as in tropical epiphytes and in some aquatic macrophytes. This water-conserving photosynthetic pathway has evolved multiple times and is found in close to 6% of vascular plant species from at least 35 families. Although many aspects of CAM molecular biology, biochemistry and ecophysiology are well understood, relatively little is known about the evolutionary origins of CAM. This review focuses on five main topics: (1) the permutations and plasticity of CAM, (2) the requirements for CAM evolution, (3) the drivers of CAM evolution, (4) the prevalence and taxonomic distribution of CAM among vascular plants with emphasis on the Orchidaceae and (5) the molecular underpinnings of CAM evolution including circadian clock regulation of gene expression.