Unique characteristics of phloem companion cells producing florigen at the single-cell level

Optimization of plant flowering timing is crucial for successful reproduction in natural environments. FLOWERING LOCUS T (FT) is a gene encoding florigen, a mobile signal that promotes flowering. The FT gene is specifically expressed in phloem companion cells in the vasculatures localized at distal parts of leaves. Despite its critical role in the transition to the reproductive stage, the difference between FT-expressing and other companion cells is still obscure. To elucidate the unique characteristics of FT-expressing cells, we applied a single-nucleus RNA-seq method using GFP-labeled nuclei sorted from target tissues. Our approach elucidated the presence of the unique cluster of companion cells with high FT transcripts. It also discovered previously unidentified phloem cells active in water transport and JA responses. Subclustering analysis against the FT-expressing cells revealed that FT expression is aligned with reduction of mesophyll cell and induction of companion cell identity, suggesting that maturation of phloem companion cells is required for FT gene expression. FT-expressing cells highly express many genes encoding small protein including anti-florigen BROTHER OF FT (BFT), raised a possibility that multiple systemic growth-regulating signals are provided from these specific companion cells. Promoters of both FT and the genes co-expressed with FT highly enriched the consensus binding sites of NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR1 (NIGT1). NIGT1.2 and NIGT1.4 significantly delayed flowering timing. Consistent with the nitrogen-dependent expression of NIGT1 genes, NIGT1s repressed FT expression especially under ample nitrogen conditions, suggesting their role as nitrogen-dependent FT repressors.