Crassulacean acid metabolism biology

Abstract

‘…As CAM genomes are completed, whole-genome sequences from all three photosynthetic types can be compared in terms of molecular evolution, structural rearrangements, and copy number variation in photosynthetic genes.’ The diurnal patterns of gas exchange and leaf acid accumulation in CAM plants contribute to CAM species' abilities to maximize biomass, CO2 uptake, and WUE under water stressed conditions. The circadian pattern of CO2 exchange in CAM plants is typically described in four phases. The first phase involves the accumulation of malic acid in the vacuole during the dark period, wherein CO2 is assimilated as a result of carbon fixation by PEPC. Phase II entails a shift from PEPC to RuBisCO activity just after dawn, followed by phase III diurnal stomatal closure, which allows for the decarboxylation of malic acid and CO2 refixation by RuBisCO. Lastly, the reopening of stomata occurs during phase IV in the evening when temperatures are lower, often resulting in a brief coupling of RuBisCO activity and stomatal opening (Davis et al., 2014). While the four phases of CAM can be discretely summarized, in nature CAM plants actually display a spectrum of phenotypes: CAM cyclers have net carbon gain during the day but recycle respired CO2 at night and accumulate malic acid; CAM idlers have a near total closure of stomata and use respired CO2 as the sole source of carbon; and facultative CAM plants can move between C3 and CAM due to environmental stressors, like salt or drought. These weak or partial forms of CAM photosynthesis have been largely understudied, as they are difficult to screen for with current methods (Pierce et al., 2002), and their position in the evolutionary trajectory of CAM is unknown. The phylogenetic status of these species, however, was brought to the forefront of the meeting in several presentations and discussions from Erika Edwards (Brown University, Providence, RI, USA), Klaus Winter (Smithsonian Tropical Research Institute, Panama City, Panama), and Katia Silvera (Smithsonian Tropical Research Institute), among others (see Winter & Holtum, 2014). Plants performing weak CAM will continue to present important opportunities to improve our understanding of the physiological and genetic gradations between C3 and CAM. A number of presenters focused on the ecology of CAM plants and the impact of climate change on the abundance and distribution of these species. Indeed, CAM plants show a large degree of environmental plasticity. Luciano Freschi (University of São Paulo, Brazil) showed how CAM expression in bromeliads is strongly influenced by both water and nutrient availability. Lonnie Guralnick (Roger Williams University, Bristol, RI, USA) spoke about the variability of CAM expression in the species Portulacaria afra and the ecological implications for interspecific variation. Cassandra Reyes Garcia (Centro de Investigación Científica de Yucatán, Mexico) discussed the colonization of trees by epiphytic bromeliads and how survival in a changing climate depends on the ability of a species to either adapt or occupy new habitats. David Williams (University of Wyoming, Laramie, WY, USA) presented work on columnar cacti in North and South America and the importance of WUE in foundation species adapted to arid climates. The CAM community has extended the drought-adapted ecophysiology of CAM plants to consideration of the potential practical applications of these species. The thick epicuticular layer found on most succulents is an ideal platform for evaluation and development of waxes and other value-added products. The high biomass production of Opuntia and Agave coupled with low water and fertilization requirements make them strong candidates for biofuel feedstocks (Borland et al., 2009). Sarah Davis (Ohio University, Athens, OH, USA) presented work from an ongoing field trial in Arizona of multiple Agave species as potential biofuel feedstocks, highlighting the photochemical and fuel-conversion efficiency of CAM plants, which is 147% greater than that of C4 species (Davis et al., 2014). Agave americana appears to be the most productive and cold-tolerant species tested, although further research will be needed to determine the economic viability of A. americana as an ethanol feedstock. Unlike Agave, Opuntia spp. has low soluble sugar content, so as processes for digestion of cellulosic materials into ethanol improve, Opuntia would be a desirable feedstock due to its low crystalline cellulose and lignin contents. Current research is also being performed to investigate the potential of inserting oil biosynthesis genes into Opuntia ficus-indica. A field trial similar to the Agave project has been started for Opuntia in southern Nevada and will be critical for determining the viability of Opuntia as a biofuel crop in the United States. The meeting also highlighted the importance of phylogenetics for understanding the evolutionary biology of CAM. While the CAM community has a number of physiological and genomic model systems (e.g. Clusia, Mesembryanthemum, Kalanchoe), phylogenetic treatments of CAM lineages are lagging. A number of lineages discussed at the meeting (e.g. Orchidaceae, Caryophyllales, Agavoideae, to name a few) are being studied phylogenetically, but many CAM groups remain under-described. Erika Edwards (Brown University) argued that when CAM is placed in a phylogenetic context, researchers have an avenue for powerful comparative analyses (Edwards & Ogburn, 2012). Comparative methods will enable the CAM community to better ascertain the number of origins of CAM, how closely related C3 and CAM species compare in terms of physiology and genetics, and whether weak forms of CAM are evolutionary stepping stones, or can persist as permanent states in some lineages. With further examination of the phylogenetic transitions between C3 and CAM, new model systems for both physiology and genomics may arise. Genomic sequencing is rapidly changing the field of plant biology as more species are sequenced and annotation progresses. While over 50 plant genomes have been published to date, none have been from CAM species. Decreases in whole-genome sequencing costs over the last few years have increased sequencing efforts and brought about draft genomes from CAM species including Agave tequilana (Oak Ridge National Laboratory, Oak Ridge, TN, USA), Ananas comosus (University of Illinois at Urbana-Champaign, IL, USA), and Kalanchoe fedtschenkoi (University of Liverpool, UK), along with genomes in Kalanchoe laxiflora (Oak Ridge National Laboratory, DOE Joint Genome Institute, Walnut Creek, CA, and University of Nevada, Reno, NV, USA) and Mesembryanthemum crystallinum (University of Nevada) currently undergoing assembly. An important area of focus will be to determine how key genes involved in photosynthesis and other genes important to CAM vary between CAM, C3 and C4 plants. As CAM genomes are completed, whole-genome sequences from all three photosynthetic types can be compared in terms of molecular evolution, structural rearrangements, and copy number variation in photosynthetic genes. A number of presentations at the symposium emphasized that the circadian nature of CAM will make transcriptomic comparisons over a diel time course critical for identifying genes that have been modified for use by CAM. Researchers who are part of the CAM Biodesign project, a multi-institution collaboration that seeks to engineer CAM into C3 biofuel feedstocks, are leading a large portion of the CAM genomics work. Global warming trends and the need for bioenergy crop production to expand into arid regions to meet future energy demands motivate this research. A comprehensive understanding of the genetic components and regulatory elements underlying CAM is vital to engineering the pathway (Borland et al., 2014). For example, Xiaohan Yang (Oak Ridge National Laboratory) discussed elucidating the genetics of CAM through comparative genomics studies of CAM, C3 and C4 plants. Comparative methods have confirmed the existence of CAM-specific genes and the expansion of gene families encoding stomatal and circadian-dependent regulatory elements. James Hartwell (University of Liverpool) described quantitative transcriptome sequencing of leaves performing C3 or CAM, in Kalanchoe fedtschenkoi, a species in which a C3 to CAM transition occurs along a developmental gradient. Hartwell's research aims to identify regulatory genes needed for CAM based on expression levels and circadian expression patterns distinct from those of C3 photosynthesis. A comparative transcriptome analysis of C3 and CAM states by Bernard Wone (University of Nevada) (Cushman et al., 2008) in M. crystallinum has revealed putative circadian-controlled CAM transcriptional regulatory networks and tentatively identifies transcription factors that may regulate key genes and phases of CAM. Research is underway to identify target genes for candidate CAM transcriptional regulators through ChIP-Seq technology. For CAM engineering, in vivo testing of putative CAM genes will be necessary to validate the CAM ‘parts list’. Susanna Boxall (University of Liverpool) described loss-of-function and gain-of-function mutants in candidate genes in Kalanchoe. Notably, phosphoenolpyruvate carboxylase kinase (PPCK) has been confirmed as necessary for nocturnal CO2 fixation and for normal circadian clock function. Analysis of CAM is also underway at the protein and metabolite levels. Paul Abraham (Oak Ridge National Laboratory) discussed how varying correlations between transcriptomic and proteomic data are most likely due to post-transcriptional and post-translational modification in A. americana. George Ratcliffe (University of Oxford, UK) detailed research on metabolic flux research in CAM. Through the modeling of CAM metabolic networks, the functional dynamics of CAM's enzymatic machinery can be more comprehensively characterized. John Cushman (University of Nevada) discussed CAM metabolomics in M. crystallinum, in which CAM-deficient mutants exhibit impaired accumulation of numerous circadian clock-controlled metabolites and starch. In addition to projects attempting to engineer the CAM pathway into common C3 crops, the tools are now in place to begin investigating changes that could be made to CAM crops to make them more agriculturally desirable. Posters and presentations at the conference reviewed several CAM species for which there are already established transformation protocols. The 34th New Phytologist meeting on CAM photosynthesis showcased the multiple approaches researchers are taking to understand and utilize CAM, from ecophysiology to evolutionary biology to functional genomics. Such an interdisciplinary approach is a natural extension of the current collaborative research environment in CAM, and many speakers highlighted the value of merging ecophysiological and genetic approaches toward the same goal. Future research should continue to use interdisciplinary methods, as CAM photosynthesis is a complex trait and will require a systems biological approach to fully describe. Tandem omics, phylogenetic, and physiological studies with existing and new models, including a number of weak or facultative CAM species, will further our understanding of the CAM spectrum and the practical applications of CAM plants.