Thorn specification in citrus plants by an SHI/STY family transcription factor

Thorns are modified branches that have evolved independently multiple times as defenses against herbivores. We previously identified the TCP transcription factors THORN IDENTITY1 (TI1) and TI2 as key regulators of thorn development in Citrus; however, how these genes are regulated remains unclear. In this study, using comparative transcriptomics, we identified TI3, encoding a SHORT INTERNODES/STYLISH (SHI/STY) family transcription factor that is specifically expressed in thorns. We found that TI3 binds to a previously undefined CTAG core element in the promoters of TI1 and TI2, activating their expression to promote stem cell arrest in the thorn meristem. CRISPR-Cas9-mediated disruption of TI3 function converted thorns into branches. Conversely, the PEBP family protein CsCENTRORADIALIS (CsCEN) represses TI3 expression in the axillary meristem to maintain stem cell activity and promote branch development. Mutations in CsCEN resulted in branch-to-thorn conversions, whereas cscen ti3 double mutants exhibited the ti3 mutant phenotype, supporting the idea that CsCEN regulates TI3 expression. The thorn-specific expression pattern of TI3 homologs across three Rutaceae species suggests that TI3 might have a conserved role in thorn development. Thus, TI3 represents a new regulator of meristem identity, and manipulating its activity is a promising approach for breeding thornless cultivars.