The Madagascan succulent plants Kalanchoë laxiflora and K. fedtschenkoi have adapted to growing in a semi-arid, sub-tropical environment in part through evolving a modified pathway of photosynthetic CO2 fixation known as Crassulacean acid metabolism (CAM) [1,2]. CAM species increase water use efficiency by opening their stomata for primary CO2 fixation in the dark, when it is cooler and more humid, and closing their stomata in the light, when the atmosphere is at its hottest and driest. The circadian clock underpins optimised temporal regulation of the pathway, preventing futile cycling between conflicting metabolic steps. Primary nocturnal CO2 fixation is catalyzed by phosphoenolpyruvate carboxylase (PPC). Temporal control of PPC is achieved via protein phosphorylation, which is catalysed by a specific circadian clock controlled protein kinase, named PPCK1.
In the Hartwell lab in Liverpool, our main goal is to identify and characterise functionally all of the genes required for the establishment and regulation of CAM [1,2]. To facilitate this, the genomes and CAM-associated transcriptomes of several members of the genus Kalanchoë have been decoded and catalogued. Candidate CAM genes are being tested by generating RNAi and/ or constitutive over-expressor transgenic lines. Examples will be presented to highlight our recent progress towards defining functionally each key component of the CAM genetic blueprint. In particular, unpublished phenotypic data for transgenic Kalanchoë lines lacking the CAM-specific PPC and PPCK1 isogenes will be presented. These lines have revealed unexpected changes in the central circadian clock and thus provide the first evidence for feedback communication between CAM-associated metabolites and the core oscillator.
We have also developed transgenic lines to test the function of a range of candidate CAM-induced and clock-controlled transcription factors (TFs) that were initially identified from RNA-seq time course datasets. Here our aim is to determine whether the candidate TFs are essential for the transmission of temporal information from the core clock to CAM. Whilst many TF lines displayed only subtle phenotypes, the down-regulation of one particular TF led to a large reduction in nocturnal CO2 fixation, and widespread down-regulation of virtually all known CAM genes. Highlights from this exciting recent breakthrough will also be presented.
 Hartwell J, Dever LV and Boxall SF (2016) Emerging model systems for functional genomics analysis of Crassulacean acid metabolism. Current Opinion in Plant Biology. 31: 100 - 108. (link is external)
 Dever LV, Boxall SF, Knerova J and Hartwell J (2015) Transgenic perturbation of the decarboxylation phase of crassulacean acid metabolism alters physiology and metabolism but has only a small effect on growth. Plant Physiology. 167: 44 - 59.