Evolution Of Crassulacean Acid Metabolism Research Articles

CAM evolution is associated with gene family expansion in an explosive bromeliad radiation.

The subgenus Tillandsia (Bromeliaceae) belongs to one of the fastest radiating clades in the plant kingdom and is characterized by the repeated evolution of Crassulacean acid metabolism (CAM). Despite its complex genetic basis, this water-conserving trait has evolved independently across many plant families and is regarded as a key innovation trait and driver of ecological diversification in Bromeliaceae. By producing high-quality genome assemblies of a Tillandsia species pair displaying divergent photosynthetic phenotypes, and combining genome-wide investigations of synteny, transposable element (TE) dynamics, sequence evolution, gene family evolution, and temporal differential expression, we were able to pinpoint the genomic drivers of CAM evolution in Tillandsia. Several large-scale rearrangements associated with karyotype changes between the 2 genomes and a highly dynamic TE landscape shaped the genomes of Tillandsia. However, our analyses show that rewiring of photosynthetic metabolism is mainly obtained through regulatory evolution rather than coding sequence evolution, as CAM-related genes are differentially expressed across a 24-h cycle between the 2 species but are not candidates of positive selection. Gene orthology analyses reveal that CAM-related gene families manifesting differential expression underwent accelerated gene family expansion in the constitutive CAM species, further supporting the view of gene family evolution as a driver of CAM evolution.

The Cissus quadrangularis genome reveals its adaptive features in an arid habitat.

Cissus quadrangularis is a tetraploid species belonging to the Vitaceae family and is known for the Crassulacean acid metabolism (CAM) pathway in the succulent stem, while the leaves perform C3 photosynthesis. Here, we report a high-quality genome of C. quadrangularis comprising a total size of 679.2Mb which was phased into two subgenomes. Genome annotation identified 51 857 protein-coding genes, while approximately 47.75% of the genome was composed of repetitive sequences. Gene expression ratios of two subgenomes demonstrated that the sub-A genome as the dominant subgenome played a vital role during the drought tolerance. Genome divergence analysis suggests that the tetraploidization event occurred around 8.9million years ago. Transcriptome data revealed that pathways related to cutin, suberine, and wax metabolism were enriched in the stem during drought treatment, suggesting that these genes contributed to the drought adaption. Additionally, a subset of CAM-related genes displayed diurnal expression patterns in the succulent stems but not in leaves, indicating that stem-biased expression of existing genes contributed to the CAM evolution. Our findings provide insights into the mechanisms of drought adaptation and photosynthesis transition in plants.

Predicting photosynthetic pathway from anatomy using machine learning.

Plants with Crassulacean acid metabolism (CAM) have long been associated with a specialized anatomy, including succulence and thick photosynthetic tissues. Firm, quantitative boundaries between non-CAM and CAM plants have yet to be established - if they indeed exist. Using novel computer vision software to measure anatomy, we combined new measurements with published data across flowering plants. We then used machine learning and phylogenetic comparative methods to investigate relationships between CAM and anatomy. We found significant differences in photosynthetic tissue anatomy between plants with differing CAM phenotypes. Machine learning-based classification was over 95% accurate in differentiating CAM from non-CAM anatomy, and had over 70% recall of distinct CAM phenotypes. Phylogenetic least squares regression and threshold analyses revealed that CAM evolution was significantly correlated with increased mesophyll cell size, thicker leaves, and decreased intercellular airspace. Our findings suggest that machine learning may be used to aid the discovery of new CAM species and that the evolutionary trajectory from non-CAM to strong, obligate CAM requires continual anatomical specialization.

Engineering Crassulacean Acid Metabolism in C3 and C4 Plants.

Carbon dioxide (CO2) is a major greenhouse gas contributing to changing climatic conditions, which is a grand challenge affecting the security of food, energy, and environment. Photosynthesis plays the central role in plant-based CO2 reduction. Plants performing CAM (crassulacean acid metabolism) photosynthesis have a much higher water use efficiency than those performing C3 or C4 photosynthesis. Therefore, there is a great potential for engineering CAM in C3 or C4 crops to enhance food/biomass production and carbon sequestration on arid, semiarid, abandoned, or marginal lands. Recent progresses in CAM plant genomics and evolution research, along with new advances in plant biotechnology, have provided a solid foundation for bioengineering to convert C3/C4 plants into CAM plants. Here, we first discuss the potential strategies for CAM engineering based on our current understanding of CAM evolution. Then we describe the technical approaches for engineering CAM in C3 and C4 plants, with a focus on an iterative four-step pipeline: (1) designing gene modules, (2) building the gene modules and transforming them into target plants, (3) testing the engineered plants through an integration of molecular biology, biochemistry, metabolism, and physiological approaches, and (4) learning to inform the next round of CAM engineering. Finally, we discuss the challenges and future opportunities for fully realizing the potential of CAM engineering.

The CAM lineages of planet Earth.

The growth of experimental studies of crassulacean acid metabolism (CAM) in diverse plant clades, coupled with recent advances in molecular systematics, presents an opportunity to re-assess the phylogenetic distribution and diversity of species capable of CAM. It has been more than two decades since the last comprehensive lists of CAM taxa were published, and an updated survey of the occurrence and distribution of CAM taxa is needed to facilitate and guide future CAM research. We aimed to survey the phylogenetic distribution of these taxa, their diverse morphology, physiology and ecology, and the likely number of evolutionary origins of CAM based on currently known lineages. We found direct evidence (in the form of experimental or field observations of gas exchange, day-night fluctuations in organic acids, carbon isotope ratios and enzymatic activity) for CAM in 370 genera of vascular plants, representing 38 families. Further assumptions about the frequency of CAM species in CAM clades and the distribution of CAM in the Cactaceae and Crassulaceae bring the currently estimated number of CAM-capable species to nearly 7 % of all vascular plants. The phylogenetic distribution of these taxa suggests a minimum of 66 independent origins of CAM in vascular plants, possibly with dozens more. To achieve further insight into CAM origins, there is a need for more extensive and systematic surveys of previously unstudied lineages, particularly in living material to identify low-level CAM activity, and for denser sampling to increase phylogenetic resolution in CAM-evolving clades. This should allow further progress in understanding the functional significance of this pathway by integration with studies on the evolution and genomics of CAM in its many forms.

Defining Mechanisms of C3 to CAM Photosynthesis Transition toward Enhancing Crop Stress Resilience.

Global climate change and population growth are persistently posing threats to natural resources (e.g., freshwater) and agricultural production. Crassulacean acid metabolism (CAM) evolved from C3 photosynthesis as an adaptive form of photosynthesis in hot and arid regions. It features the nocturnal opening of stomata for CO2 assimilation, diurnal closure of stomata for water conservation, and high water-use efficiency. To cope with global climate challenges, the CAM mechanism has attracted renewed attention. Facultative CAM is a specialized form of CAM that normally employs C3 or C4 photosynthesis but can shift to CAM under stress conditions. It not only serves as a model for studying the molecular mechanisms underlying the CAM evolution, but also provides a plausible solution for creating stress-resilient crops with facultative CAM traits. This review mainly discusses the recent research effort in defining the C3 to CAM transition of facultative CAM plants, and highlights challenges and future directions in this important research area with great application potential.

The climate changes promoted the chloroplast genomic evolution of Dendrobium orchids among multiple photosynthetic pathways

Dendrobium orchids have multiple photosynthetic pathways, which can be used as a model system for studying the evolution of crassulacean acid metabolism (CAM). In this study, based on the results of the net photosynthetic rates (Pn), we classified Dendrobium species into three photosynthetic pathways, then employed and compared their chloroplast genomes. The Dendrobium chloroplast genomes have typical quartile structures, ranging from 150,841–153,038 bp. The apparent differences in GC content, sequence variability, and IR junctions of SSC/IRB junctions (JSBs) were measured within chloroplast genomes among different photosynthetic pathways. The phylogenetic analysis has revealed multiple independent CAM origins among the selected Dendrobium species. After counting insertions and deletions (InDels), we found that the occurrence rates and distribution densities among different photosynthetic pathways were inconsistent. Moreover, the evolution patterns of chloroplast genes in Dendrobium among three photosynthetic pathways were also diversified. Considering the diversified genome structure variations and the evolution patterns of protein-coding genes among Dendrobium species, we proposed that the evolution of the chloroplast genomes was disproportional among different photosynthetic pathways. Furthermore, climatic correlation revealed that temperature and precipitation have influenced the distribution among different photosynthetic pathways and promoted the foundation of CAM pathway in Dendrobium orchids. Based on our study, we provided not only new insights into the CAM evolution of Dendrobium but also provided beneficial genetic data resources for the further systematical study of Dendrobium.

Evolution of Crassulacean acid metabolism in response to the environment: past, present, and future

Crassulacean acid metabolism (CAM) is a mode of photosynthesis that evolved in response to decreasing CO2 levels in the atmosphere some 20 million years ago. An elevated ratio of O2 relative to CO2 caused many plants to face increasing stress from photorespiration, a process exacerbated for plants living under high temperatures or in water-limited environments. Today, our climate is again rapidly changing and plants’ ability to cope with and adapt to these novel environments is critical for their success. This review focuses on CAM plant responses to abiotic stressors likely to dominate in our changing climate: increasing CO2 levels, increasing temperatures, and greater variability in drought. Empirical studies that have assessed CAM responses are reviewed, though notably these are concentrated in relatively few CAM lineages. Other aspects of CAM biology, including the effects of abiotic stress on the light reactions and the role of leaf succulence, are also considered in the context of climate change. Finally, more recent studies using genomic techniques are discussed to link physiological changes in CAM plants with the underlying molecular mechanism. Together, the body of work reviewed suggests that CAM plants will continue to thrive in certain environments under elevated CO2. However, how CO2 interacts with other environmental factors, how those interactions affect CAM plants, and whether all CAM plants will be equally affected remain outstanding questions regarding the evolution of CAM on a changing planet.

CAM Models: Lessons and Implications for CAM Evolution.

The evolution of Crassulacean acid metabolism (CAM) by plants has been one of the most successful strategies in response to aridity. On the onset of climate change, expanding the use of water efficient crops and engineering higher water use efficiency into C3 and C4 crops constitute a plausible solution for the problems of agriculture in hotter and drier environments. A firm understanding of CAM is thus crucial for the development of agricultural responses to climate change. Computational models on CAM can contribute significantly to this understanding. Two types of models have been used so far. Early CAM models based on ordinary differential equations (ODE) reproduced the typical diel CAM features with a minimal set of components and investigated endogenous day/night rhythmicity. This line of research brought to light the preponderant role of vacuolar malate accumulation in diel rhythms. A second wave of CAM models used flux balance analysis (FBA) to better understand the role of CO2 uptake in flux distribution. They showed that flux distributions resembling CAM metabolism emerge upon constraining CO2 uptake by the system. We discuss the evolutionary implications of this and also how CAM components from unrelated pathways could have integrated along evolution.

Differential timing of gene expression and recruitment in independent origins of CAM in the Agavoideae (Asparagaceae).

Crassulacean acid metabolism (CAM) photosynthesis has evolved repeatedly across the plant tree of life, however our understanding of the genetic convergence across independent origins remains hampered by the lack of comparative studies. Here, we explore gene expression profiles in eight species from the Agavoideae (Asparagaceae) encompassing three independent origins of CAM. Using comparative physiology and transcriptomics, we examined the variable modes of CAM in this subfamily and the changes in gene expression across time of day and between well watered and drought-stressed treatments. We further assessed gene expression and the molecular evolution of genes encoding phosphoenolpyruvate carboxylase (PPC), an enzyme required for primary carbon fixation in CAM. Most time-of-day expression profiles are largely conserved across all eight species and suggest that large perturbations to the central clock are not required for CAM evolution. By contrast, transcriptional response to drought is highly lineage specific. Yucca and Beschorneria have CAM-like expression of PPC2, a copy of PPC that has never been shown to be recruited for CAM in angiosperms. Together the physiological and transcriptomic comparison of closely related C3 and CAM species reveals similar gene expression profiles, with the notable exception of differential recruitment of carboxylase enzymes for CAM function.

Underwater CAM photosynthesis elucidated by Isoetes genome

To conserve water in arid environments, numerous plant lineages have independently evolved Crassulacean Acid Metabolism (CAM). Interestingly, Isoetes, an aquatic lycophyte, can also perform CAM as an adaptation to low CO2 availability underwater. However, little is known about the evolution of CAM in aquatic plants and the lack of genomic data has hindered comparison between aquatic and terrestrial CAM. Here, we investigate underwater CAM in Isoetes taiwanensis by generating a high-quality genome assembly and RNA-seq time course. Despite broad similarities between CAM in Isoetes and terrestrial angiosperms, we identify several key differences. Notably, Isoetes may have recruited the lesser-known ‘bacterial-type’ PEPC, along with the ‘plant-type’ exclusively used in other CAM and C4 plants for carboxylation of PEP. Furthermore, we find that circadian control of key CAM pathway genes has diverged considerably in Isoetes relative to flowering plants. This suggests the existence of more evolutionary paths to CAM than previously recognized.

Evolution of crassulacean acid metabolism (CAM) as an escape from ecological niche conservatism in Malagasy Bulbophyllum (Orchidaceae).

Despite growing evidence that niche shifts are more common in flowering plants than previously thought, little is known of whether such shifts are promoted by changes in photosynthetic pathways. Here we combine the most complete phylogeny for epiphytic Malagasy Bulbophyllum orchids (c.210 spp.) with climatic niche and carbon isotope ratios to infer the group's spatial-temporal history, and the role of strongly expressed crassulacean acid metabolism (CAM) in facilitating niche shifts and diversification. We find that most extant species still retain niche (Central Highland) and photosynthesis (C3 ) states as present in the single mid-Miocene (c.12.70million yr ago (Ma)) ancestor colonizing Madagascar. However, we also infer a major transition to CAM, linked to a late Miocene (c.7.36Ma) invasion of species from the sub-humid highland first into the island's humid eastern coastal, and then into the seasonally dry 'Northwest Sambirano' rainforests, yet without significant effect on diversification rates. These findings indicate that CAM in tropical epiphytes may be selectively advantageous even in high rainfall habitats, rather than presenting a mere adaptation to dry environments or epiphytism per se. Overall, our study qualifies CAM as an evolutionary 'gateway' trait that considerably widened the spatial-ecological amplitude of Madagascar's most species-rich orchid genus.

Engineering of Crassulacean Acid Metabolism.

Crassulacean acid metabolism (CAM) has evolved from a C3 ground state to increase water use efficiency of photosynthesis. During CAM evolution, selective pressures altered the abundance and expression patterns of C3 genes and their regulators to enable the trait. The circadian pattern of CO2 fixation and the stomatal opening pattern observed in CAM can be explained largely with a regulatory architecture already present in C3 plants. The metabolic CAM cycle relies on enzymes and transporters that exist in C3 plants and requires tight regulatory control to avoid futile cycles between carboxylation and decarboxylation. Ecological observations and modeling point to mesophyll conductance as a major factor during CAM evolution. The present state of knowledge enables suggestions for genes for a minimal CAM cycle for proof-of-concept engineering, assuming altered regulation of starch synthesis and degradation are not critical elements of CAM photosynthesis and sufficient malic acid export from the vacuole is possible.

The eco-evolutionary significance of rainfall constancy for facultative CAM photosynthesis.

A flexible use of the crassulacean acid metabolism (CAM) has been hypothesised to represent an intermediate stage along a C3 to full CAM evolutionary continuum, when relative contributions of C3 vs CAM metabolism are co-determined by evolutionary history and prevailing environmental constraints. However, evidence for such eco-evolutionary interdependencies is lacking. We studied these interdependencies for the leaf-succulent genus Drosanthemum (Aizoaceae, Southern African Succulent Karoo) by testing for relationships between leaf δ13 C diagnostic for CAM dependence (i.e. contribution of C3 and CAM to net carbon gain), and climatic variables related to temperature and precipitation and their temporal variation. We further quantified the effects of shared phylogenetic ancestry on CAM dependence and its relation to climate. CAM dependence is predicted by rainfall and its temporal variation, with high predictive power of rainfall constancy (temporal entropy). The predictive power of rainfall seasonality and temperature-related variables was negligible. Evolutionary history of the tested clades significantly affected the relationship between rainfall constancy and CAM dependence. We argue that higher CAM dependence might provide an adaptive advantage in increasingly unpredictable rainfall environments when the anatomic exaptation (succulence) is already present. These observations might shed light on the evolution of full CAM.

Metabolic Modeling of the C3-CAM Continuum Revealed the Establishment of a Starch/Sugar-Malate Cycle in CAM Evolution.

The evolution of Crassulacean acid metabolism (CAM) is thought to be along a C3-CAM continuum including multiple variations of CAM such as CAM cycling and CAM idling. Here, we applied large-scale constraint-based modeling to investigate the metabolism and energetics of plants operating in C3, CAM, CAM cycling, and CAM idling. Our modeling results suggested that CAM cycling and CAM idling could be potential evolutionary intermediates in CAM evolution by establishing a starch/sugar-malate cycle. Our model analysis showed that by varying CO2 exchange during the light period, as a proxy of stomatal conductance, there exists a C3-CAM continuum with gradual metabolic changes, supporting the notion that evolution of CAM from C3 could occur solely through incremental changes in metabolic fluxes. Along the C3-CAM continuum, our model predicted changes in metabolic fluxes not only through the starch/sugar-malate cycle that is involved in CAM photosynthetic CO2 fixation but also other metabolic processes including the mitochondrial electron transport chain and the tricarboxylate acid cycle at night. These predictions could guide engineering efforts in introducing CAM into C3 crops for improved water use efficiency.

Leaf anatomy is not correlated to CAM function in a C3+CAM hybrid species, Yucca gloriosa.

Crassulacean acid metabolism (CAM) is often considered to be a complex trait, requiring orchestration of leaf anatomy and physiology for optimal performance. However, the observation of trait correlations is based largely on comparisons between C3 and strong CAM species, resulting in a lack of understanding as to how such traits evolve and the level of intraspecific variability for CAM and associated traits. To understand intraspecific variation for traits underlying CAM and how these traits might assemble over evolutionary time, we conducted detailed time course physiological screens and measured aspects of leaf anatomy in 24 genotypes of a C3+CAM hybrid species, Yucca gloriosa (Asparagaceae). Comparisons were made to Y. gloriosa's progenitor species, Y. filamentosa (C3) and Y. aloifolia (CAM). Based on gas exchange and measurement of leaf acids, Y. gloriosa appears to use both C3 and CAM, and varies across genotypes in the degree to which CAM can be upregulated under drought stress. While correlations between leaf anatomy and physiology exist when testing across all three Yucca species, such correlations break down at the species level in Y. gloriosa. The variation in CAM upregulation in Y. gloriosa is a result of its relatively recent hybrid origin. The lack of trait correlations between anatomy and physiology within Y. gloriosa indicate that the evolution of CAM, at least initially, can proceed through a wide combination of anatomical traits, and more favourable combinations are eventually selected for in strong CAM plants.

The Evolution of CAM Photosynthesis in Australian Calandrinia Reveals Lability in C3+CAM Phenotypes and a Possible Constraint to the Evolution of Strong CAM.

Australian Calandrinia has radiated across the Australian continent during the last 30 Ma, and today inhabits most Australian ecosystems. Given its biogeographic range and reports of facultative Crassulacean acid metabolism (CAM) photosynthesis in multiple species, we hypothesized (1) that CAM would be widespread across Australian Calandrinia and that species, especially those that live in arid regions, would engage in strong CAM, and (2) that Australian Calandrinia would be an important lineage for informing on the CAM evolutionary trajectory. We cultivated 22 Australian Calandrinia species for a drought experiment. Using physiological measurements and δ13C values we characterized photosynthetic mode across these species, mapped the resulting character states onto a phylogeny, and characterized the climatic envelopes of species in their native ranges. Most species primarily utilize C3 photosynthesis, with CAM operating secondarily, often upregulated following drought. Several phylogenetically nested species are C3, indicating evolutionary losses of CAM. No strong CAM was detected in any of the species. Results highlight the limitations of δ13C surveys in detecting C3+CAM phenotypes, and the evolutionary lability of C3+CAM phenotypes. We propose a model of CAM evolution that allows for lability and reversibility among C3+CAM phenotypes and C3 and suggest that an annual life-cycle may preclude the evolution of strong CAM.

Shared expression of crassulacean acid metabolism (CAM) genes pre-dates the origin of CAM in the genus Yucca.

Crassulacean acid metabolism (CAM) is a carbon-concentrating mechanism that has evolved numerous times across flowering plants and is thought to be an adaptation to water-limited environments. CAM has been investigated from physiological and biochemical perspectives, but little is known about how plants evolve from C3 to CAM at the genetic or metabolic level. Here we take a comparative approach in analyzing time-course data of C3, CAM, and C3+CAM intermediate Yucca (Asparagaceae) species. RNA samples were collected over a 24 h period from both well-watered and drought-stressed plants, and were clustered based on time-dependent expression patterns. Metabolomic data reveal differences in carbohydrate metabolism and antioxidant response between the CAM and C3 species, suggesting that changes to metabolic pathways are important for CAM evolution and function. However, all three species share expression profiles of canonical CAM pathway genes, regardless of photosynthetic pathway. Despite differences in transcript and metabolite profiles between the C3 and CAM species, shared time-structured expression of CAM genes in both CAM and C3Yucca species suggests that ancestral expression patterns required for CAM may have pre-dated its origin in Yucca.

Operating at the very low end of the crassulacean acid metabolism spectrum: Sesuvium portulacastrum (Aizoaceae).

Demonstration of crassulacean acid metabolism (CAM) in species with low usage of this system relative to C3-photosynthetic CO2 assimilation can be challenging experimentally but provides crucial information on the early steps of CAM evolution. Here, weakly expressed CAM was detected in the well-known pantropical coastal, leaf-succulent herb Sesuvium portulacastrum, demonstrating that CAM is present in the Sesuvioideae, the only sub-family of the Aizoaceae in which it had not yet been shown conclusively. In outdoor plots in Panama, leaves and stems of S. portulacastrum consistently exhibited a small degree of nocturnal acidification which, in leaves, increased during the dry season. In potted plants, nocturnal acidification was mainly facultative, as levels of acidification increased in a reversible manner following the imposition of short-term water-stress. In drought-stressed plants, nocturnal net CO2 exchange approached the CO2-compensation point, consistent with low rates of CO2 dark fixation sufficient to eliminate respiratory carbon loss. Detection of low-level CAM in S. portulacastrum adds to the growing number of species that cannot be considered C3 plants sensu stricto, although they obtain CO2 principally via the C3 pathway. Knowledge about the presence/absence of low-level CAM is critical when assessing trajectories of CAM evolution in lineages. The genus Sesuvium is of particular interest because it also contains C4 species.

Conservation and Diversification of Circadian Rhythmicity Between a Model Crassulacean Acid Metabolism Plant Kalanchoë fedtschenkoi and a Model C3 Photosynthesis Plant Arabidopsis thaliana.

Crassulacean acid metabolism (CAM) improves photosynthetic efficiency under limited water availability relative to C3 photosynthesis. It is widely accepted that CAM plants have evolved from C3 plants and it is hypothesized that CAM is under the control of the internal circadian clock. However, the role that the circadian clock plays in the evolution of CAM is not well understood. To identify the molecular basis of circadian control over CAM evolution, rhythmic gene sets were identified in a CAM model plant species (Kalanchoë fedtschenkoi) and a C3 model plant species (Arabidopsis thaliana) through analysis of diel time-course gene expression data using multiple periodicity detection algorithms. Based on protein sequences, ortholog groups were constructed containing genes from each of these two species. The ortholog groups were categorized into five gene sets based on conservation and diversification of rhythmic gene expression. Interestingly, minimal functional overlap was observed when comparing the rhythmic gene sets of each species. Specifcally, metabolic processes were enriched in the gene set under circadian control in K. fedtschenkoi and numerous genes were found to have retained or gained rhythmic expression in K. fedtsechenkoi. Additonally, several rhythmic orthologs, including CAM-related orthologs, displayed phase shifts between species. Results of this analysis point to several mechanisms by which the circadian clock plays a role in the evolution of CAM. These genes provide a set of testable hypotheses for future experiments.