Tissue-and cell-type-specific gene expression in cotyledons and true leaves. (A) Representative images of ClearSee-treated cotyledons and true leaves of pFT:NTF, pSUC2:NTF, pCAB2:NTF and p35:NTF lines. Scale bar, 500 µm. (B) The first two principal components of bulk RNA-seq analysis for three independent cotyledon and true leaf samples are described in (A). Cotyledon and true leaf samples are circled in blue and red, respectively. (C) DEseq2-normalized counts of FT transcripts in sorted nuclei for the pFT:NFT, pSUC2:NFT, and pCAB2:NFT lines compared to the p35S:NTF line. Fold enrichments of FT transcripts in each NTF line compared with those in p35S:NTF line are indicated. ***padj<0.001. (D–K) Expression of genes encoding CO stabilizers (D and E), FT activators (F and G), CO destabilizers (H and I), and FT repressors (J and K) in cotyledons (D, F, H and J) and true leaves (E, G, I and K). Genes encoding proteins belonging to the same family were clustered in the heatmap. Bar color indicates log2-scaled fold-change relative to the p35S:NTF line. Asterisks denote significant differences from p35S:NTF (*padj<0.05; **padj <0.01; ***padj <0.001). CO was removed from the analysis due to insufficient reads at ZT4.

Single-nuclei RNA-seq identifies distinct subpopulations of phloem companion cells. (A) UMAP with the origin of nuclei indicated by color. (B and C) Table with the number of nuclei originated from each line and sample (B) and median UMI and number of genes detected per nucleus (C). (D) Seurat defined clusters with cluster numbers indicated. (E–G) UMAP annotated with normalized read counts for FT (E), SUC2 (F), and FLP1 (G) expression. (H–I) UMAP annotated with average read counts of genes related to ATP biosynthesis (H), water transport (I), and JA responses (J). Color bars indicate gene expression levels. For the lists of genes, see Fig. S7D (pink highlighted), S8E and S9B.

Phloem companion cell and mesophyll cell marker gene expression in cluster 7. (A) Subclustering of cluster 7. Colors indicate each subcluster. (B) Table showing the number of nuclei originated from each NTF line in cluster 7. (C) UMAP of normalized read counts of companion cell marker genes (FT, SUC2, and AHA3) and mesophyll cell marker genes (RBCS1A, CAB3, and CAB2). (D) Violin plot of normalized read counts of marker genes for companion cells and mesophyll cells across the three subclusters.

FT-expressing cells express genes encoding other small proteins. (A) Amino acid length of proteins encoded by genes differentially expressed in clusters 4, 5, and 7. Black bars indicate the median amino acid length. (B) Expression of the pFT:NFT line (green) and promoter fusions of selected cluster 7 genes with H2B-tdTomato (red) in true leaves. The selected genes were differentially expressed in cluster 7 and encoded small proteins. Yellow color shows an overlap between green and red signals. Scale bar, 50 µm. (C) Flowering time measurements of T1 transgenic plants overexpressing selected cluster 7 genes driven by the pSUC2 promoter. Eight genes were tested, five of which were tested for overlap with FT expression in (B).

NIGT1 transcription factors are repressors of FT. (A) Motif enrichment analysis using the 268 genes differentially expressed in cluster 7 and flowering time of the pSUC2:NIGT1.2 and pSUC2:NIGT1.4 lines. The NIGT1.2 binding site was the most enriched motif in cluster 7 differentially expressed genes. The bottom and top lines of the box indicate the first and third quantiles, and the bottom and top lines of the whisker denote minimum and maximum values. The bar inside the box is the median value (n =16). (B) A Venn diagram showing the significantly down-regulated genes from bulk RNA-seq of two independent pSUC2:NIGT1.2 and pSUC2:NIGT1.4 lines. (C) Relative gene expression levels of 14-day old WT and nigtQ seedlings at ZT4. Plants were grown with high nitrogen (+N) and without (–N). Asterisks denote significant differences from WT (*P<0.05, **P<0.01, ***P<0.001, t-test). n.s. indicates not significant.