Transcriptional analysis of flower and thorn development in Citrus

Although both thorns and flowers originate from determinate shoot meristems, they develop into morphologically distinct structures with different biological functions. However, the molecular mechanisms underlying their early developmental divergence remain largely unknown. In this study, we performed comparative transcriptomic analysis of young thorns, young flowers, and adjacent stems from citrange trees to investigate the regulatory programs associated with these two determinate shoot types. We identified 480 flower-preferential and 400 thorn-preferential genes. Flower-preferential genes include canonical MADS-box genes involved in flower development (e.g., APETALA3 ( CsAP3 ), PISTILLATA ( CsPI ), AGAMOUS ( CsAG ), and SEPALLATA3 ( CsSEP3 )), the floral meristem identity gene LEAFY ( CsLFY ), and known regulators of stamen (e.g., DYSFUNCTIONAL TAPETUM 1 ( CsDYT1 ) and ABORTED MICROSPORES ( CsAMS )) and fruit development (e.g., CRABS CLAW ( CsCRC )). Thorn-preferential genes include the thorn identity genes TI1 , TI2 , and TI3 , along with the thorn tip sclerification regulatory factor SHORT AND SOFT THORN 1 ( SST1 ) and its downstream targets (e.g., NAC secondary wall thickening promoting factor 1 ( CsNST1 ) and secondary wall-associated NAC domain protein 1 ( CsSND1 )). Surprisingly, the A-class flower development genes CsAP1 and CsAP2.1 were preferentially expressed in young thorns, and several MADS-box genes, including SHORT VEGETATIVE PHASE ( SVP ) homologs and SUPPRESSOR OF OVEREXPRESSION OF CO 1 ( SOC1 )-like genes, also exhibited thorn-preferential expression. Functional analysis using CRISPR-Cas9-mediated knockout demonstrated that loss of CsAP1 resulted in significantly shortened thorns, indicating a role for this floral identity gene in thorn growth. Collectively, our findings provide new insights into the developmental programs of thorns and flowers and offer candidate genes for targeted modification of these shoot types in Citrus .