Stomatal Responses to Light, CO2, and Mesophyll Tissue in Vicia faba and Kalanchoë fedtschenkoi

The responses of stomatal aperture to light intensity and CO2 concentration were studied in both Vicia faba (C3) and Kalanchoë fedtschenkoi (Crassulacean acid metabolism; CAM), in material sampled from both light and dark periods. Direct comparison was made between intact leaf segments, epidermises grafted onto exposed mesophyll, and isolated epidermal peels, including transplantations between species and between diel periods. We reported the stomatal opening in response to darkness in isolated CAM peels from the light period, but not from the dark. Furthermore, we showed that C3 mesophyll has stimulated CAM stomata in transplanted peels to behave as C3 in response to light and CO2. By using peels and mesophyll from plants sampled in the dark and the light period, we provided clear evidence that CAM stomata behaved differently from C3. This might be linked to stored metabolites/ions and signalling pathway components within the guard cells, and/or a mesophyll-derived signal. Overall, our results provided evidence for both the involvement of guard cell metabolism and mesophyll signals in stomatal responses in both C3 and CAM species.

Inference of Gene Regulatory Network Uncovers the Linkage between Circadian Clock and Crassulacean Acid Metabolism in Kalanchoë fedtschenkoi

The circadian clock drives time-specific gene expression, enabling biological processes to be temporally controlled. Plants that conduct crassulacean acid metabolism (CAM) photosynthesis represent an interesting case of circadian regulation of gene expression as stomatal movement is temporally inverted relative to stomatal movement in C3 plants. The mechanisms behind how the circadian clock enabled physiological differences at the molecular level is not well understood. Recently, the rescheduling of gene expression was reported as a mechanism to explain how CAM evolved from C3. Therefore, we investigated whether core circadian clock genes in CAM plants were re-phased during evolution, or whether networks of phase-specific genes were simply re-wired to different core clock genes. We identified candidate core clock genes based on gene expression features and then applied the Local Edge Machine (LEM) algorithm to infer regulatory relationships between this new set of core candidates and known core clock genes in Kalanchoë fedtschenkoi. We further inferred stomata-related gene targets for known and candidate core clock genes and constructed a gene regulatory network for core clock and stomata-related genes. Our results provide new insight into the mechanism of circadian control of CAM-related genes in K. fedtschenkoi, facilitating the engineering of CAM machinery into non-CAM plants for sustainable crop production in water-limited environments.

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. Expected final online publication date for the Annual Review of Plant Biology, Volume 72 is May 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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.