A promoter::luciferase reporter gene imaging toolkit in Kalanchoë laxiflora reveals molecular elements responsible for the circadian regulation of Crassulacean acid metabolism (CAM)

Crassulacean acid metabolism (CAM) plants perform primary atmospheric CO ₂ fixation at night, with timekeeping by the endogenous circadian clock. Understanding of circadian coordination of CAM remains limited to rhythmic post-translational regulation of phosphoenolpyruvate carboxylase (PPC) by a specific clock-controlled protein kinase, PPCK. Here, candidate promoter regions (∼3000 bp) of CAM-associated genes from Kalanchoë laxiflora were coupled to a firefly luciferase reporter and stable transgenic lines of both K. laxiflora and C ₃ Arabidopsis thaliana were generated. In K. laxiflora, the CAM-associated GLUCOSE 6-PHOSPHATE/PHOSPHATE TRANSLOCATOR2 promoter ( KlGPT2p ) generated robust circadian rhythms of luciferase luminescence in constant conditions, with peak activity in leaf pair 3, where CAM-associated nocturnal CO ₂ fixation initiated during leaf development. KlGPT2p::LUC+ did not drive rhythms of luminescence in A. thaliana and the KlPPCK1 promoter produced no LUC+ signal in either species. Furthermore, the CHLOROPHYLL A/B BINDING PROTEIN2 promoter ( KlCAB2p ), a clock-controlled promoter that drives a gene involved in light-reactions of photosynthesis, drove robust rhythms in both K. laxiflora and A. thaliana . KlCAB2p circadian period changed during leaf development in K. laxiflora, revealing differing control by the core-clock during development. KlCAB2p peak activity shifted to dawn in A. thaliana relative to a dusk phased peak in CAM leaves of K. laxiflora , highlighting differences in the timing of outputs from the core clock between species. These findings establish a robust PROMOTER::LUC+ reporter system in a CAM plant and highlight divergent timing driving clock controlled promoters between species, and period lengthening with leaf age in Kalanchoë .