CYP722A1-mediated 16-hydroxylation of carlactonoic acid regulates the floral transition in <i>Arabidopsis</i>

Strigolactones (SLs) are multifunctional plant hormones and rhizosphere signals with diverse structures, roughly classified into two categories: canonical and noncanonical SLs. In Arabidopsis thaliana, SL biosynthesis mutants exhibit increased shoot branching and early flowering, underscoring their roles in developmental regulation. Shoot branching inhibition in Arabidopsis is associated with the methylation of a noncanonical SL, carlactonoic acid (CLA), catalyzed by CLA methyltransferase (CLAMT). Canonical SLs primarily function as rhizosphere signals, with their biosynthesis in dicots mediated by CYP722C enzymes. It is hypothesized that Arabidopsis does not produce canonical SL because of the lack of the CYP722C genes in its genome. Instead, Arabidopsis possesses CYP722A1, a member of the previously uncharacterized CYP722A subfamily, distinct from the CYP722C subfamily. This study demonstrates that Arabidopsis cyp722a1 mutants exhibit an earlier floral transition without excessive shoot branching. Biochemical analysis revealed that CYP722A1 catalyzes the hydroxylation of CLA to produce 16-hydroxy-CLA (16-HO-CLA), which is subsequently methylated by CLAMT to form 16-HO-MeCLA. 16-HO-CLA and 16-HO-MeCLA were detected in the wildtype; however, these compounds were absent in max1-4 mutant, deficient in CLA synthesis, and in cyp722a1 mutant. These findings show CYP722A1-dependent 16-hydroxylation activity of CLA in Arabidopsis. Moreover, they suggest that hydroxylated CLA specifically regulates floral transition, distinct from branching inhibition. Through the identification of CYP722A1 affecting floral transition, which is the distinct role of the CYP722A subfamily, this work provides insights into the structural diversification of SLs for specialized biological functions in plant development.