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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 CO 2 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. Despite extensive characterization of crassulacean acid metabolism (CAM) in terrestrial angiosperms, little attention has been given to aquatics and early diverging land plants. Here, the authors assemble the genome of Isoetes taiwanensis and investigate the genetic factors driving CAM in this aquatic lycophyte.

Crassulacean acid metabolism (CAM), one of three kinds of photosynthesis, is a water-use efficient adaptation to an arid environment. CAM is characterized by CO2 uptake via open stomata during the nighttime and refixation CO2 via the Calvin cycle during the daytime. Facultative CAM plants can shift the photosynthesis from C3 to CAM and exhibit greater plasticity in CAM expression under different environments. Though leaf thickness is an important anatomical feature of CAM plants, there may be no anatomical feature changes during the C3–CAM transition for all facultative CAM plants. The shift from C3 photosynthesis to CAM in facultative CAM plants is accompanied by significant changes in physiology including stomata opening, CO2 gas exchange and organic acid fluxes; the activities of many decarboxylating enzymes increase during the shift from C3 to CAM; the molecular changes occur during the photosynthesis C3–CAM shift involved DNA hypermethylation, transcriptional regulation, post-transcriptional regulation and protein level regulation. Recently, omics approaches were used to discover more proceedings underling the C3–CAM transition. However, there are few reviews on the mechanisms involved in this photosynthetic shift in facultative CAM plants. In this paper, we summarize the progress in the comparative analysis of anatomical, physiological, metabolic and molecular properties of facultative CAM plants between C3 and CAM photosynthesis. Facultative CAM plants also show the potential for sustainable food crop and biomass production. We also discuss the implications of the photosynthesis transition from C3 to CAM on horticultural crops and address future directions for research.

Societal Impact Statement Research efforts in plant biology have often been focused on sequenced and well‐studied ‘model’ organisms. Despite the advent of relatively inexpensive genome sequencing, most plant taxonomic groups are underrepresented, with few species that ‘represent’ the diversity of whole genera. This study describes an economical guide to sequencing a non‐model organism, which may be useful in reducing the cost of sequencing more species within genera and across plant life. This method was used to develop Kalanchoë blossfeldiana as a resource for comparing C3 and the water‐conserving mode of photosynthesis known as Crassulacean acid metabolism (CAM) within the same plant. Summary Despite the increasing number of well‐studied plant species with well‐annotated genomes across plant life, there are few densely sampled genera with more than a couple of genome sequences representing the diversity of whole genera. Here, we develop an economic approach to full‐genome sequencing that could be used to sequence many species within a genus. We made use of the Nanopore rapid sequencing kit to assist in plant genome assembly, dramatically reducing the cost. Here we applied this method to cost‐effectively develop genomic resources for Kalanchoë blossfeldiana, a commercially important ornamental, in which Crassulacean Acid Metabolism (CAM), a water‐conserving mode of photosynthesis can be induced. We present a physiological and biochemical characterisation of Kalanchoe blossfeldiana with its nuclear and chloroplastic genome and a comparative C3, CAM dusk transcriptome. We apply this approach to a complex tetraploid genome, making use of a relative species for chromosomal scaffolding to reduce assembly ploidy, we provide a resource for future gene expression studies. We highlight its limitations, e.g. the need for deeper sequencing to accurately resolve genome structure and haplotypes without using a relative species for scaffolding. The study demonstrates the merits of K. blossfeldiana as a comparative system for studying C3 and CAM within a plant and has identified substantial changes in the dusk transcriptome between young C3 and mature CAM K. blossfeldiana leaves in response to age‐induced CAM, and shows that in the absence of abiotic stress, CAM induction still involves the engagement of drought and abscisic acid (ABA) response pathways. Research efforts in plant biology have often been focused on sequenced and well‐studied ‘model’ organisms. Despite the advent of relatively inexpensive genome sequencing, most plant taxonomic groups are underrepresented, with few species that ‘represent’ the diversity of whole genera. This study describes an economical guide to sequencing a non‐model organism, which may be useful in reducing the cost of sequencing more species within genera and across plant life. This method was used to develop Kalanchoë blossfeldiana as a resource for comparing C3 and the water‐conserving mode of photosynthesis known as Crassulacean acid metabolism (CAM) within the same plant.

Are evolutionary outcomes predictable? Adaptations that show repeated evolutionary convergence across the Tree of Life provide a special opportunity to dissect the context surrounding their origins, and identify any commonalities that may predict why certain traits evolved many times in particular clades and yet never evolved in others. The remarkable convergence of C₄ and Crassulacean Acid Metabolism (CAM) photosynthesis in vascular plants makes them exceptional model systems for understanding the repeated evolution of complex phenotypes. This review highlights what we have learned about the recurring assembly of C₄ and CAM, focusing on the increasingly predictable stepwise evolutionary integration of anatomy and biochemistry. With the caveat that we currently understand C₄ evolution better than we do CAM, I propose a general model that explains and unites C₄ and CAM evolutionary trajectories. Available data suggest that anatomical modifications are the ‘rate-limiting step’ in each trajectory, which in large part determines the evolutionary accessibility of both syndromes. The idea that organismal structure exerts a primary influence on innovation is discussed in the context of other systems. Whether the rate-limiting step occurs early or late in the evolutionary assembly of a new phenotype may have profound implications for its distribution across the Tree of Life.

Stable carbon isotope ratios (δ13C) of terrestrial plants are employed across a diverse range of applications in environmental and plant sciences; however, the kind of information that is desired from the δ13C signal often differs. At the extremes, it ranges between purely environmental and purely biological. Here, we review environmental drivers of variation in carbon isotope discrimination (Δ) in terrestrial plants, and the biological processes that can either damp or amplify the response. For C3 plants, where Δ is primarily controlled by the ratio of intercellular to ambient CO2 concentrations (c i/c a), coordination between stomatal conductance and photo-synthesis and leaf area adjustment tends to constrain the potential environmentally driven range of Δ. For C4 plants, variation in bundle-sheath leakiness to CO2 can either damp or amplify the effects of c i/c a on Δ. For plants with crassulacean acid metabolism (CAM), Δ varies over a relatively large range as a function of the proportion of daytime to night-time CO2 fixation. This range can be substantially broadened by environmental effects on Δ when carbon uptake takes place primarily during the day. The effective use of Δ across its full range of applications will require a holistic view of the interplay between environmental control and physiological modulation of the environmental signal.

Unlike C₃ plants, Crassulacean acid metabolism (CAM) plants fix CO₂ in the dark using phosphoenolpyruvate carboxylase (PPC; EC 4.1.1.31). PPC combines phosphoenolpyruvate with CO₂ (as HCO₃⁻), forming oxaloacetate. The oxaloacetate is converted to malate, leading to malic acid accumulation in the vacuole, which peaks at dawn. During the light period, malate decarboxylation concentrates CO₂ around Rubisco for secondary fixation. CAM mutants lacking PPC have not been described. Here, we employed RNA interference to silence the CAM isogene PPC1 in Kalanchoë laxiflora. Line rPPC1-B lacked PPC1 transcripts, PPC activity, dark period CO₂ fixation, and nocturnal malate accumulation. Light period stomatal closure was also perturbed, and the plants displayed reduced but detectable dark period stomatal conductance and arrhythmia of the CAM CO₂ fixation circadian rhythm under constant light and temperature free-running conditions. By contrast, the rhythm of delayed fluorescence was enhanced in plants lacking PPC1. Furthermore, a subset of gene transcripts within the central circadian oscillator was upregulated and oscillated robustly in this line. The regulation of guard cell genes involved in controlling stomatal movements was also perturbed in rPPC1-B. These findings provide direct evidence that the regulatory patterns of key guard cell signaling genes are linked with the characteristic inverse pattern of stomatal opening and closing during CAM.

Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that features nocturnal CO2 uptake, facilitates increased water-use efficiency (WUE), and enables CAM plants to inhabit water-limited environments such as semi-arid deserts or seasonally dry forests. Human population growth and global climate change now present challenges for agricultural production systems to increase food, feed, forage, fiber, and fuel production. One approach to meet these challenges is to increase reliance on CAM crops, such as Agave and Opuntia, for biomass production on semi-arid, abandoned, marginal, or degraded agricultural lands. Major research efforts are now underway to assess the productivity of CAM crop species and to harness the WUE of CAM by engineering this pathway into existing food, feed, and bioenergy crops. An improved understanding of CAM has potential for high returns on research investment. To exploit the potential of CAM crops and CAM bioengineering, it will be necessary to elucidate the evolution, genomic features, and regulatory mechanisms of CAM. Field trials and predictive models will be required to assess the productivity of CAM crops, while new synthetic biology approaches need to be developed for CAM engineering. Infrastructure will be needed for CAM model systems, field trials, mutant collections, and data management.

Portulaca grandiflora simultaneously utilizes both the C4 and Crassulacean acid metabolism (CAM) photosynthetic pathways. Our goal was to determine whether CAM developed and was functional simultaneously with the C4 pathway in cotyledons of P. grandiflora. We studied during development whether CAM would be induced with water stress by monitoring the enzyme activity, leaf structure, JO2 (rate of O2 evolution calculated by fluorescence analysis), and the changes in titratable acidity of 10 and 25 days old cotyledons. In the 10 days old cotyledons, C4 and CAM anatomy were evident within the leaf tissue. The cotyledons showed high titratable acid levels but a small CAM induction. In the 25 days old cotyledons, there was a significant acid fluctuation under 7 days of water stress. The overall enzyme activity was reduced in the 10 days old plants, while in the 25 days old plants CAM activity increased under water-stressed conditions. In addition to CAM, the research showed the presence of glycine decarboxylase in the CAM tissue. Thus, it appears both pathways develop simultaneously in the cotyledons but the CAM pathway, due to anatomical constraints, may be slower to develop than the C4 pathway. Cotyledons showed the ancestral Atriplicoid leaf anatomy, which leads to the question: Could a CAM cell be the precursor to the C4 pathway? Further study of this may lead to understanding into the evolution of C4 photosynthesis in the Portulaca.

• 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 spatialtemporal 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. 12.70 million yr ago (Ma)) ancestor colonizing Madagascar. However, we also infer a major transition to CAM, linked to a late Miocene (c. 7.36 Ma) 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.

Phalaenopsis orchids exhibit remarkable photosynthetic plasticity, enabling them to effectively acclimate crassulacean acid metabolism (CAM) to a wide range of light levels. Herein, the photosynthetic acclimation of Phalaenopsis Queen Beer ‘Mantefon’ was examined under different light intensities. Phalaenopsis clones grown under a photosynthetic photon flux density (PPFD) of 100 µmol m −2  s −1 were subjected to different light intensities of 10, 50, 100, and 200 µmol m −2  s −1 for either one day or two months of modified light levels, and their chlorophyll fluorescence response and CO 2 exchange rate were observed. The electron transport rate (ETR) varied rapidly to changing light levels, showing a significant positive correlation with light intensity after just one day of treatment. Only plants exposed to an elevated light intensity of 200 µmol m −2  s −1 for 1 day showed a decrease in ETR after midday. Moreover, after 2 months, the ETR decreased more slowly under 200 µmol m −2  s −1 . Long-term exposure to varying light conditions for two months led to increased CO 2 uptake, even at reduced light intensities. The plants also exhibited an enhanced malic acid recovery rate under both low- and high-light conditions. Citric acid levels also varied with light intensity. High-light conditions led to a significant increase in plant growth, characterized by greater biomass and a higher number of leaves. Furthermore, stable carbon isotope analysis revealed differences in the daytime CO 2 uptake rate of Phalaenopsis plants grown under different light intensities for two months. In this manner, Phalaenopsis orchids exhibit remarkable plasticity in their photosynthetic pathways, allowing them to acclimate effectively to different light environments. Investigating Phalaenopsis light acclimation is crucial for understanding the mechanisms underlying photosynthetic optimization and growth in diverse light environments in CAM plants.