Negative feedback regulation of karrikin signaling in Arabidopsis thaliana by an antagonistic paralog of karrikin receptors

Karrikins (KARs) are a class of butenolide molecules discovered in smoke hypothesized to mimic an undiscovered plant hormone, KAI2 ligand (KL). KAR/KL signaling regulates germination, seedling development, stress tolerance, and symbiotic interactions with soil microbes, among other traits. KAR/KL signaling is initiated by KARRIKIN INSENSITIVE2 (KAI2), an ɑ/β-hydrolase related to the strigolactone enzyme-receptor DWARF14 (D14). Activated KAI2 forms protein–protein interactions that trigger proteasomal degradation of a transcriptional regulator, SUPPRESSOR OF MAX2 1 (SMAX1), initiating changes in gene expression. D14-LIKE2 ( DLK2 ), an ancient paralog of KAI2 and D14 , is a prominent transcriptional marker of KAR/KL signaling in many plants that has uncertain function. We find that DLK2 forms a negative feedback loop that attenuates KAR/KL signaling in Arabidopsis thaliana . This mechanism complements that of KARRIKIN UPREGULATED F-BOX1 ( KUF1 ), which putatively restricts KAR/KL metabolism through targeted protein degradation. Loss-of-function mutations of DLK2 show little effect alone, but synthetically enhance the constitutive KAR/KL responses of kuf1 seedlings. Overexpression of DLK2 proteins from several plants increases the abundance of a SMAX1 ratiometric reporter. DLK2 does not require nuclear localization to protect SMAX1, suggesting its function is independent of interactions with SMAX1 or its transcriptional regulator partners. DLK2 hydrolyzes a profluorescent, desmethyl butenolide reporter molecule that is putatively analogous to KL. We hypothesize that DLK2 catabolizes KAI2 ligand(s) without participating in KAR/KL signaling directly. This functional antagonism could have evolved after KAI2 gene duplication through subfunctionalizing mutations that disrupted protein–protein interactions while preserving enzymatic activity.