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 CO2 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 Kalanchoe laxiflora were coupled to a firefly luciferase reporter and stable transgenic lines of both K. laxiflora and C3 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 CO2 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 Kalanchoe. One-sentence SummaryRobust circadian rhythms of firefly luciferase in the Crassulacean acid metabolism (CAM) model species Kalanchoe laxiflora were driven by both CAM and non-CAM gene promoters.