Figure 4.

LBDs negatively regulate cytokinin signaling and promote cell growth during secondary growth

(A) Heatmap showing normalized log₂FoldChange (FC) of A-type ARRs mRNA in LBD3 or LBD11 inducible overexpression RNA-seq data. We considered a gene “not expressed” in mature root if read counts were less than 10.

(B) qRT-PCR analysis of A-type ARR (ARR5 and ARR15) transcription in 9-day-old roots (0.5–2 cm below the root-hypocotyl junction, undergoing secondary growth) with 8 h mock or induction. Data are presented as mean ± SE from three biological replicates. Two-tailed t test. ^∗p < 0.05; ^∗∗p < 0.01.

(C and D) Confocal microscopy of ARR5_pro:erYFP (C) and TCSn_pro:nYFP (D) after 1-day LBD3 induction in 6-day-old roots. ARR5_pro:erYFP was analyzed in F1 generation.

(E and F) Confocal microscopy of ARR5_pro:erYFP (E) and TCSn_pro:nYFP (F) in 7-day-old WT and lbd3;4 roots.

(G) Quantification of average fluorescent signal intensity in (E) and (F). Area marked with brackets (E and F) was quantified. Red dots indicate average fluorescent signal intensity in individual roots. n, number of independent roots analyzed.

(H) Cross-sections of 5-day-old roots of LBD inducible overexpression lines. Three-day-old roots were treated for 2 days with mock or 5 μM 17-β, except in the case of LBD3, which was treated with 0.5 μM 17-β. Green dots represent pericycle cells. Cells inside of dotted line were used for cell file quantification in Figure S4G.

(I) Cross-sections of 14-day-old roots of LBD inducible overexpression lines. Eight-day-old roots were treated for 6 days with mock or 5 μM 17-β. Note that 0.5 μM 17-β was used for the LBD3 hemizygous (hem) line due to dose-dependent effect (Figure S4M).

(J) A model presenting the roles of LBDs during the progression of root secondary growth (large blue arrow) downstream of cytokinin (CK).

Dashed lines represent root boundaries (C–F). Primary xylem cells (i.e., xylem axis) are false colored in blue (H and I). Scale bars, 100 μm (C–F), 20 μm (H), and 50 μm (I). See also Figures S1G and S4, Table S2, and Data S1 and S3.