A PROMOTER::LUCIFERASE reporter system reveals key elements of the circadian regulation of Crassulacean acid metabolism (CAM) in Kalanchoë laxiflora Baker

Jun 5, 2026The Plant journal : for cell and molecular biology

Key parts controlling daily rhythms of water-saving metabolism in Kalanchoë laxiflora revealed by a gene activity reporter

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Abstract

The promoter region of the CAM-associated gene KlGPT2p generated robust circadian rhythms in Kalanchoë laxiflora.

KlGPT2p was linked to a luciferase reporter, producing peak luminescence activity in leaf pair 3 of K. laxiflora.
No luminescence rhythms were observed in Arabidopsis with KlGPT2p, indicating species-specific differences in promoter function.
The KlCAB2p promoter drove strong circadian rhythms in both K. laxiflora and Arabidopsis, showing its effectiveness across species.
Circadian period for KlCAB2p varied during leaf development in K. laxiflora, with peak activity shifting from dusk to dawn between species.
These results suggest diverse timing mechanisms in clock-controlled gene expression in CAM plants compared to non-CAM species.

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Crassulacean acid metabolism (CAM) plants perform primary atmospheric COfixation 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 Baker were coupled to a firefly luciferase reporter and stable transgenic lines of both K. laxiflora and CArabidopsis 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 COfixation initiated during leaf development. KlGPT2p::LUC+ did not drive rhythms of luminescence in Arabidopsis 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 Arabidopsis. KlCAB2p circadian period changed during leaf development in K. laxiflora, and peak activity shifted from dusk in CAM leaves of K. laxiflora to dawn in Arabidopsis, 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, highlight divergent timing driving clock-controlled promoters between species, and period lengthening with leaf age in Kalanchoë. 2 3 2

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A PROMOTER::LUCIFERASE reporter system reveals key elements of the circadian regulation of Crassulacean acid metabolism (CAM) in Kalanchoë laxiflora Baker

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