Following publication of the original article.

Already a proven mechanism for drought resilience, crassulacean acid metabolism (CAM) is a specialized type of photosynthesis that maximizes water-use efficiency by means of an inverse (compared to C₃ and C₄ photosynthesis) day/night pattern of stomatal closure/opening to shift CO₂ uptake to the night, when evapotranspiration rates are low. A systems-level understanding of temporal molecular and metabolic controls is needed to define the cellular behaviour underpinning CAM. Here, we report high-resolution temporal behaviours of transcript, protein and metabolite abundances across a CAM diel cycle and, where applicable, compare the observations to the well-established C₃ model plant Arabidopsis. A mechanistic finding that emerged is that CAM operates with a diel redox poise that is shifted relative to that in Arabidopsis. Moreover, we identify widespread rescheduled expression of genes associated with signal transduction mechanisms that regulate stomatal opening/closing. Controlled production and degradation of transcripts and proteins represents a timing mechanism by which to regulate cellular function, yet knowledge of how this molecular timekeeping regulates CAM is unknown. Here, we provide new insights into complex post-transcriptional and -translational hierarchies that govern CAM in Agave. These data sets provide a resource to inform efforts to engineer more efficient CAM traits into economically valuable C₃ crops.

Background

Crassulacean acid metabolism (CAM), a specialized mode of photosynthesis, enables plant adaptation to water-limited environments and improves photosynthetic efficiency via an inorganic carbon-concentrating mechanism. Kalanchoë fedtschenkoi is an obligate CAM model featuring a relatively small genome and easy stable transformation. However, the molecular responses to light quality and intensity in CAM plants remain understudied.

Results

Here we present a genome-wide expression atlas of K. fedtschenkoi plants grown under 12 h/12 h photoperiod with different light quality (blue, red, far-red, white light) and intensity (0, 150, 440, and 1,000 μmol m-2 s-1) based on RNA sequencing performed for mature leaf samples collected at dawn (2 h before the light period) and dusk (2 h before the dark period). An eFP web browser was created for easy access of the gene expression data. Based on the expression atlas, we constructed a light-responsive co-expression network to reveal the potential regulatory relationships in K. fedtschenkoi. Measurements of leaf titratable acidity, soluble sugar, and starch turnover provided metabolic indicators of the magnitude of CAM under the different light treatments and were used to provide biological context for the expression dataset. Furthermore, CAM-related subnetworks were highlighted to showcase genes relevant to CAM pathway, circadian clock, and stomatal movement. In comparison with white light, monochrome blue/red/far-red light treatments repressed the expression of several CAM-related genes at dusk, along with a major reduction in acid accumulation. Increasing light intensity from an intermediate level (440 μmol m-2 s-1) of white light to a high light treatment (1,000 μmol m-2 s-1) increased expression of several genes involved in dark CO2 fixation and malate transport at dawn, along with an increase in organic acid accumulation.

Conclusions

This study provides a useful genomics resource for investigating the molecular mechanism underlying the light regulation of physiology and metabolism in CAM plants. Our results support the hypothesis that both light intensity and light quality can modulate the CAM pathway through regulation of CAM-related genes in K. fedtschenkoi.

Background

Crassulacean acid metabolism (CAM) enhances plant water-use efficiency through an inverse day/night pattern of stomatal closure/opening that facilitates nocturnal CO₂ uptake. CAM has evolved independently in over 35 plant lineages, accounting for ~ 6% of all higher plants. Agave species are highly heat- and drought-tolerant, and have been domesticated as model CAM crops for beverage, fiber, and biofuel production in semi-arid and arid regions. However, the genomic basis of evolutionary innovation of CAM in genus Agave is largely unknown.

Results

Using an approach that integrated genomics, gene co-expression networks, comparative genomics and protein structure analyses, we investigated the molecular evolution of CAM as exemplified in Agave. Comparative genomics analyses among C₃, C₄ and CAM species revealed that core metabolic components required for CAM have ancient genomic origins traceable to non-vascular plants while regulatory proteins required for diel re-programming of metabolism have a more recent origin shared among C₃, C₄ and CAM species. We showed that accelerated evolution of key functional domains in proteins responsible for primary metabolism and signaling, together with a diel re-programming of the transcription of genes involved in carbon fixation, carbohydrate processing, redox homeostasis, and circadian control is required for the evolution of CAM in Agave. Furthermore, we highlighted the potential candidates contributing to the adaptation of CAM functional modules.

Conclusions

This work provides evidence of adaptive evolution of CAM related pathways. We showed that the core metabolic components required for CAM are shared by non-vascular plants, but regulatory proteins involved in re-reprogramming of carbon fixation and metabolite transportation appeared more recently. We propose that the accelerated evolution of key proteins together with a diel re-programming of gene expression were required for CAM evolution from C₃ ancestors in Agave.

Crassulacean acid metabolism (CAM) is a water-use efficient adaptation of photosynthesis that has evolved independently many times in diverse lineages of flowering plants. We hypothesize that convergent evolution of protein sequence and temporal gene expression underpins the independent emergences of CAM from C₃ photosynthesis. To test this hypothesis, we generate a de novo genome assembly and genome-wide transcript expression data for Kalanchoë fedtschenkoi, an obligate CAM species within the core eudicots with a relatively small genome (~260 Mb). Our comparative analyses identify signatures of convergence in protein sequence and re-scheduling of diel transcript expression of genes involved in nocturnal CO₂ fixation, stomatal movement, heat tolerance, circadian clock, and carbohydrate metabolism in K. fedtschenkoi and other CAM species in comparison with non-CAM species. These findings provide new insights into molecular convergence and building blocks of CAM and will facilitate CAM-into-C₃ photosynthesis engineering to enhance water-use efficiency in crops.

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.