A single nucleotide polymorphism in the 4-Coumarate CoA ligase 1-likeα promoter induces juice sac granulation by promoting brassinolide-mediated lignin synthesis in citrus

Juice sac granulation [also known as "Kushui" {KS}], characterized by hardening and dehydration, is a widespread physiological disorder that severely compromises fruit quality. However, the molecular mechanism and signaling pathways underlying this disorder remain largely unexplored. In this study, we identified a unique hybrid from a cross between Kiyomi (Citrus unshiu Marc × Citrus sinensis Osbeck) and Egan No. 1 tangerine (Citrus reticulata Blanc.) that exhibits the juice sac granulation from the onset of fruit development. Compared to normal fruits, "KS" fruit exhibited significantly elevated levels of lignin and brassinolide (BR). Through resequencing and transcriptome analyses, we identified 4-Coumarate CoA ligase 1-likeα (Ci4CL1-likeα) as a key gene involved in lignin biosynthesis. Notably, a single nucleotide polymorphism (SNP, A/T) in the promoter region of Ci4CL1-likeα between KS (carrying T) and normal (carrying A) allowed the former to be successfully targeted and positively regulated by CiMYB306. In addition, both CiMYB306 and Ci4CL1-likeα were positively regulated by CiNAC9 through interacting with the NARC motifs in a BR-inducible way. Genetic evidences confirmed that CiMYB306 and CiNAC9 regulated lignin biosynthesis in a Ci4CL1-likeα-dependent manner. Taken together, our findings reveal that the mutation of A into T in the promoter of Ci4CL1-likeα creates a binding site recognized and positively activated by CiMYB306, which acts in synergy with CiNAC9 in a BR-responsive way, leading to enhanced lignin synthesis and fruit granulation. These findings gain valuable insight into the BR-mediated transcriptional regulatory module involved in fruit granulation and unravel potential targets for mitigating this disorder.