Crassulacean acid metabolism (CAM) at the crossroads: a special issue to honour 50 years of CAM research by Klaus Winter

A spectacular feature of the botanical world is the succulent flora, which presents a diversity of life forms from the fascinating to the strange (Figs 1–3). Succulence describes fleshy tissue with high water content, yet as a character state is highly variable, ranging from slightly fleshy planar leaves to the grossly corpulent stems of cacti and aloes (Fig. 2; Grace, 2019; Peréz- Lopéz et al., 2023). The associated function also shows marked variation, ranging from water and salt storage, mechanical support and, in the majority of succulents, to the storage of organic acids used in crassulacean acid metabolism (CAM). Succulence is hypothesized to be essential for the function of the CAM photosynthetic pathway because it allows storage of large amounts of assimilated carbon in the form of malate, and assists the CAM process by trapping CO2 released during the day (Males, 2017; Borland et al., 2018; Edwards, 2019). In the typical form of CAM photosynthesis, plants open stomata at night to fix inorganic carbon into malic acid using phosphoenolpyruvate (PEP) carboxylase (see figs 1 and 2 in Chomthong and Griffiths, 2023, in this issue for explanatory diagrams). The malic acid is then stored in large vacuoles. During the day, stomata close, and the stored malic acid is decarboxylated to raise CO2 concentrations within leaves and stems to levels that suppress the wasteful process of photorespiration. Rubisco then refixes the released CO2 that is trapped within the leaves by the closed stomata, and the fixation products are synthesized into sugars and starch by the same biochemistry used in C3 photosynthesis. Because stomata are closed over much of the day, transpiration is low and thus CAM plants exhibit high water-use efficiencies (WUE) of photosynthesis. High WUE enables survival in dry locations, to include arid and semi-arid landscapes, and soil-less environments such as the epiphyte habitat on tree branches or the lithophyte habit on rock faces. A montage showing some of the morphological diversity of CAM succulents evident along roadsides of southern Africa in April 2016. (A) Tylocodon wallichi – Crassulaceae (photo courtesy of Matt Stata); (B) Adromischus spp., possibly A. alstonii – Crassulaceae; (C) Conophytum bilobum – Aizoaceae; (D) Larryleachia spp., possibly L. marlothii – Apocynaceae (barrel milkweed); (E) Anacampseros papyracea – Anacampserotaceae; (F) Lithops spp., possibly L. julii – Aizoaceae; (I) Conophytum spp., possibly C. praesectum – Aizoaceae; (I) Mesembryanthemum spp., possibly M. crystallinum – Aizoaceae; (J) Mesembryanthemum, possibly M. guerichianum – Aizoaceae. All photos by R. F. Sage except where noted. CAM is abundant and well dispersed across the world’s flora occurring in ferns, gymnosperms, monocots and eudicots. As examined in this issue (Gilman et al., 2023), CAM photosynthesis independently evolved over 60 times across vascular plants, making it one of the best examples of evolutionary convergence on a complex trait in the living world. The repeated evolution of CAM has resulted in hundreds of genera and close to 18 000 species that exhibit some degree of CAM, with numerous species being recognized as the iconic plants of the world’s arid landscapes and epiphytic habitats, for example as shown by the saguaro and prickly-pear cacti, agaves, aloes, epiphytic orchids, succulent euphorbs and Spanish moss (Figs 2 and 3). CAM is thus considered a key adaptation that helped establish the vegetation of drier habitats of the modern biosphere. For humans, CAM plants help support livelihoods in semi-arid climates, and for many more, the diverse forms of CAM vegetation bring beauty and inspiration in natural habitats and gardens, and as indoor succulents. Iconic CAM plants of the world. (A) Saguaro cactus (Carnegiea gigantea) near Tuscon, Arizona, USA; (B) spiral aloe (Aloe polyphylla, at Seascapes Succulent Nursery, Half Moon Bay, CA, USA) (photo taken with the permission of Janice Moody, owner of Seascapes Nursery); (C) Agave species at the UNAM Botanical Garden, Mexico City, Mexico (photo by K. Heyduk); (D) Spanish moss (Tillandsia usneoides, at Saint Marks, National Wildlife Refuge, FL, USA); (E) Vanilla pomposa, in cultivation in Panama (photo courtesy of Katia Silvera). Photos by R. F. Sage except where indicated otherwise. Three iconic CAM species representing the potential for exploiting CAM in an agroforestry setting on semi-arid and often degraded lands. In A, invasive Opuntia ficus-indica infesting dry, marginal landscapes near Marysdale in central South Africa, demonstrating the potential of this genus to be a productive bioenergy crop on semi-arid landscapes with minimal agronomic inputs. In B, the cactus Lophocereus schottii (old man cactus) grows well on the harsh landscape of southern Baja California, Mexico. In C, a stem succulent Euphorbia virosa grows well in the arid landscape of the Namib desert in west-central Namibia. Euphorbia species are considered promising biofuel species because their latex can be refined into motor fuels. Panels B and C show remarkable convergent evolution onto a common form of stem succulence that is associated with CAM diversification in these two unrelated clades, both of which evolved from weakly succulent C3 ancestors. Photos by R. F. Sage. Since the elucidation of the CAM biochemical cycle, scholars have pondered its evolutionary origins and why it has evolved so frequently (Evans, 1971; Kluge and Ting, 1978; Ehleringer and Monson, 1993). With advances in phylogenomics, evolutionary research has accelerated, bringing new understanding to how, why and where CAM evolved, and when it evolved (Edwards, 2019, 2023; Heyduk, 2022; Sage et al., 2023). This research is exploiting the deep foundation of CAM knowledge built up over decades by physiologists, ecologists and anatomists, such that the diversity of CAM across the Earth is at least well appreciated, if not completely understood (Holtum, 2023b). On a separate front, exploiting CAM crops is now seen as a means of expanding agricultural production into non-arable lands, due to their tolerance of severe drought, anthropogenic degradation or enhanced salinity (Borland et al., 2009; Pereira et al., 2021). Moreover, many CAM plants are highly plastic, showing an ability to switch between C3 and strong CAM photosynthetic modes (Dodd et al., 2002; Borland et al., 2011). In an agronomic context, this ability allows CAM plants to use a C3 photosynthetic mode when water is abundant to realize high growth rates, and when drought intensifies, to rely on the CAM mode to survive (Borland et al., 2011). In a changing climate with greater frequencies of heat and drought stress, such flexibility could allow CAM crops to survive and maintain productivity in dry, marginal landscapes. In a world of increasing demand for agricultural products, CAM promises to make a significant contribution to global crop productivity, particularly if advances in molecular biology enable the introduction of the CAM pathway into existing C3 and C4 crops (Borland et al., 2014; Lim et al., 2019; Pereira et al., 2021). As with many species across the Earth, CAM plants are also threatened by the multiple ways in which humans are altering the global environment (Sage and Stata, 2021; Hultine et al., 2023). Epiphytic CAM species that comprise the majority of the global CAM flora are particularly endangered due to widespread loss of forested land in the tropics and subtropics (D’Antonio and Vitousek, 1992; Lewis et al., 2015; Zotz et al., 2023). Populations of arid-zone CAM plants such as the iconic cacti of the Americas are also being degraded by runaway fire cycles, climate extremes, infestation by invasive species and overharvesting for the horticultural trade (Hultine et al., 2023). Cacti show some of the highest heat tolerance thresholds recorded in vascular plants (Downton et al., 1984), but this ability could be both a blessing and a curse. Their high tolerance may pre-adapt them to increasing aridity as climates warm, but ironically, heat-tolerant organisms often exist near maximum limits of viability and for this reason are vulnerable to even modest climate change if it pushes them over their absolute viability thresholds (Somero, 2010). Thus, CAM photosynthesis and the CAM flora can be viewed as approaching a crossroads. Likewise, CAM research is also at a crossroads, made possible by a new generation of tools and techniques. These developments promise rapid advances in the understanding of CAM origins in space and time, how CAM increases fitness of natural populations, and how humanity might exploit CAM in novel ways. At the same time, the CAM flora of the Earth is imperiled by rapid acceleration of anthropogenic activities that are changing the climate, the Earth’s biogeochemistry and the cover of natural landscapes (Sage, 2020). Ironically, precisely when scientists and agronomists are in the best position to understand, appreciate and utilize CAM plants, much of the CAM flora might be lost to advancing global climate change. With the realization that the CAM flora is facing great change, the editors of Annals of Botany decided that this is an ideal time to prepare a special issue entitled CAM at the Crossroads. In organizing the special issue, we sought the contributions of multiple generations of CAM researchers, including those at the end of distinguished careers, early career scientists and many in between. In particular, we wanted to highlight the contributions of Dr Klaus Winter, a leader whose research transcends multiple generations of CAM scholars. Since his first CAM publication (Winter and von Willert, 1972), Winter has maintained a leading research programme on CAM biology, first as a PhD student in Ulrich Lüttge’s lab at the University of Darmstadt in Germany, then as a faculty member at the University of Würzburg. Since 1991, Winter has worked as a senior research scientist at the Smithsonian Tropical Research Institute in Panama City, Panama, allowing him direct access to a rich CAM flora in its natural element. His leadership in CAM goes well beyond his prolific publications, discoveries and data acquisition. The future of any field is dependent upon the engagement of colleagues in supporting and mentoring the next generation, in building networks of collegiality, and in serving as a source for conferences, reviews, books and perspectives that provide new directions while the state of the the in a is as to the of a field as is the research and so it is essential to have to provide the and to a growth and Winter has this leader for CAM biology, and because of his we can that CAM research has a thus this special issue of CAM at the to his decades of Dr Winter to that is not yet and promises to maintain his high of contributions for to The special issue of and research into the that highlight the of CAM biology and decades of CAM The issue with a of research by a close of many (Holtum, In this many of the and that made for particularly rich His the to many colleagues worked with Winter over the demonstrating that great are built by and is by a from Dr Winter in which a of his career as a his of CAM, how to his life to CAM and some of the of his at the Smithsonian Tropical Research Institute 2023). to the into the future of CAM research and on the and of a life in The CAM diversity and its future with an introduction to CAM and a on the state of CAM research and Griffiths, 2023). The promising developments for understanding CAM and their with some on the future of the CAM flora, that CAM plants are expanding in such as the of the yet are threatened by overharvesting for the horticultural with a on CAM diversity and global of CAM which the CAM and habitat diversity are for a CAM clades, with into how climate, and evolution the modern CAM et with a to the of CAM photosynthesis the vascular of the and CAM and This the first significant to the CAM of the world and Winter their in the and (Winter and and colleagues the of CAM in genera of vascular plants, representing using of for CAM, ranging from carbon and the of these their a of evolutionary origins of CAM the vascular Their also increases the of CAM species to of vascular The that this is by means but a foundation that the of CAM species and evolutionary origins in the et at the to be considered a CAM In such as CAM and CAM, and CAM to but with advances by Winter, and CAM as a of character from CAM, to strong CAM with minimal contributions from direct of CO2 the tissue stomata (Winter et al., 2015; Winter, 2019; et al., 2021). are now to many species with CAM, leading to a realization that it might be to CAM as an to C3 photosynthesis in such plants, a photosynthetic pathway as is the with plants where of the carbon is by et with an of CAM potential in the South desert This species has high carbon for C3 plants, the of C4 or CAM photosynthesis. C4 photosynthesis by has stems and is drought – which it is a typical C3 These an that many arid and yet has recognized as CAM et al., In their of photosynthesis in et it is a C3 CAM of its carbon at night CAM in the stem that while the of photosynthesis over the its contributions during drought, at which the but contributions of CAM survival on particularly dry in its arid As global CAM diversity is the of a evolutionary but how threatened is the CAM flora by the of anthropogenic global Hultine et CAM species in the future using the cacti as a multiple from global change, including direct of habitat loss from climate and land use change, and by invasive of the significant to is of due to a of climate change, invasive and drier are the of these because enable a between the of cactus The invasive form a which during the dry increases fire and of while climate and increases the of the (Sage and Stata, 2021). Cacti and succulents are of and of CAM succulents often one or two fire Moreover, are also strong that suppress cactus a In the epiphytic of the CAM plants are not as widespread as et al., 2023). epiphytic CAM species make up of the world’s CAM flora, a to many the iconic of CAM succulents in the of the and of to is epiphytic particularly in of dry where CAM epiphyte can be much have examined in epiphytic change also a to the CAM the can be complex of drier and changing an of the CAM epiphytic flora in the and, exhibit adaptation to their et future climates of habitat on and that species habitat as climate In many their that the at which habitat the habitat space for CAM The of the special issue CAM evolution and with a by that of CAM evolutionary many have on how CAM has evolved, that CAM evolution and for of the evolution of C4 photosynthesis et al., that the research might from that in CAM much the that the C4 has of C4 presents two of how a CAM might in C3 such of CAM evolution to be well and such as and have for the of understanding of C4 evolution as CAM research such a evolutionary et al., 2014; Pereira et al., 2023). The large of CAM evolutionary many for but have examined in the and the could as for CAM evolution et al., 2023; et al., 2023). et the and future of CAM research in while et a for of these has multiple origins of CAM, a in of species and character for evolutionary and species are for the rapid of generations that evolutionary research et al., a to and in the CAM The may be one such at least for C3 CAM evolution et al., species have in molecular of CAM function et al., et al., CAM evolved is an that with it for how change the evolution of photosynthetic and how CAM plants might in a world where CO2 concentrations are increasing a Sage et that CAM in the in of in CO2 that low CO2 CAM in with tissue succulence and the of and associated as climates of CAM CO2 that low CO2 the contribution of the CAM to carbon (Sage et al., 2023). that CO2 the contribution from C3 of photosynthesis, such that any of CAM be the that CAM plants in particularly harsh in high CO2 At high CO2 during drought, CAM be in carbon when stomata are completely In the Crassulaceae the of strong CAM in high CO2 is also The to the common lithophyte life form of Crassulaceae As Sage et these plants are often for rock where of could low and a CO2 in even in high CO2 CAM both from C3 and diversification of CAM its the of from species in an evolutionary is Because CAM many due to often high diversification CAM (Sage et al., 2023), this can be a but with modern and As an example of the potential of deep for evolutionary et the from species of a as a novel bioenergy crop for marginal et al., this in and possibly as a of adaptation to a of environments as the the of this in a fitness could provide new for the of Opuntia as well as understanding how evolution novel to into a new in CAM evolution is the between CAM and and succulence is a state for strong CAM function (Edwards, 2019). a et and of and succulence in the of of one at and the two at and succulence to in clades, leading the to that it a key that the to into the epiphytic also CAM could be a key that this future of CAM across the As with C4 photosynthesis, CAM is recognized as being the evolutionary of in and (Sage, 2002; with succulence being one of a of provide a of CAM et examined in and across a CAM in that the contribution of the CAM mode is with and that low transpiration from CAM a in This that to CAM function in C3 species to CAM, the vascular to be in to the of biochemical and on CAM evolution is by et the function of of photosynthetic within CAM which has to the between the of the photosynthetic tissue and to of the released CO2 and et that fitness associated with low within photosynthetic are because the in CAM leaves is from stomata and across the and not within the that might be an evolutionary and with fitness or might for such as a to for this is or it the of up with The in CAM has direct from et with support being or et al., and et might thus evolutionary of CAM and using from evolutionary of CAM photosynthesis. The of the special issue presents a diverse of and research on the of CAM species and to the and of CAM CAM species are in with a of Agave species as leading with aloe and As and Agave species have for by in the recognized the of the plants in dry in the plants that can be to CAM their the large allow storage of a of products and while that the succulent leaves provide for These have of dry of the Americas for of and provide promise for a of products in future as the crops are et understanding of Agave by how the in the Agave species and Agave The at least Agave species that now exist as within rock built by to and The living plants are of that have not by the and promise to be species for carbon in plants, be considered in to photosynthesis, because the of carbon loss and thus production as well as and Borland in CAM plants a has much carbon As a C in CAM plants could from in C3 and C4 plants because CAM species have and that could and CAM rates, a support with new data from a of of CAM in plants rates, particularly during and Borland their by of in CAM plants, such as which of CAM that of CAM into C3 crops to that are to CAM in to CAM function and carbon in the promising for are the Opuntia cacti whose and provide for in and for the world. Opuntia species are of degraded landscapes, such that have invasive species of their (Fig. et al., Opuntia plants promise to be of for biofuel particularly on marginal land in semi-arid of the world et al., 2015; et al., 2021). Opuntia is as an CAM yet C3 might help high levels of productivity of Opuntia the of CAM in and of cactus et al., 2023). In plants, the as that to CAM CO2 fixation is that CAM the means of photosynthetic carbon in these et al., 2023). In research a of CO2 In the dependent upon starch during the day, starch not to et al., 2023). These provide new into the over stomata and WUE in CAM plants that help for the CAM pathway into C3 of C3 CAM can promising for CAM et C3 CAM in C3 CAM are of et al., 2020). where early generation of CAM, the between CAM and C3 of resulted in plants that are C3 in with The that is a promising genus in which to C3 CAM the of CAM In the in this special issue, an of the developments in CAM diversity CAM CAM and CAM the special issue an for the to of the contributions of Klaus Winter, as an example of how a and leadership can a field for over and As we to the we the of CAM research high one be the and of CAM which help establish for CAM clades, the understanding of CAM in the diverse and establish of A is the elucidation of how CAM evolved and why it with high This the of of CAM the of and of how the of the CAM character state evolutionary CAM evolution have beyond CAM, because CAM is an for complex trait and convergent evolution in in because the many allow for that of CAM A research is to ways to the cultivation and of CAM crops to make use of often for agricultural production and A research is to CAM into C3 and to CAM in existing CAM These could provide an for to or agricultural while also exploiting degraded and marginal landscapes. The of CAM could to be a for to both the environment while agricultural production in the of the global climate new and ways to be to and the CAM flora for essential and where this is not CAM in locations, be botanical gardens, of succulent plants or the many succulent and the botanical of the a large to of the CAM research with from over the world is This is the best to the of Klaus Winter and his many colleagues built the foundation of CAM the next generation of these and in CAM but also be for the of the CAM flora and its to across the

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.