Brassinosteroids regulate organ boundary formation in the shoot apical meristem of Arabidopsis

Abstract

Spatiotemporal control of the formation of organ primordia and organ boundaries from the stem cell niche in the shoot apical meristem (SAM) determines the patterning and architecture of plants, but the underlying signaling mechanisms remain poorly understood. Here we show that brassinosteroids (BRs) play a key role in organ boundary formation by repressing organ boundary identity genes. BR-hypersensitive mutants display organ-fusion phenotypes, whereas BR-insensitive mutants show enhanced organ boundaries. The BR-activated transcription factor BZR1 directly represses the cup-shaped cotyledon (CUC) family of organ boundary identity genes. In WT plants, BZR1 accumulates at high levels in the nuclei of central meristem and organ primordia but at a low level in organ boundary cells to allow CUC gene expression. Activation of BR signaling represses CUC gene expression and causes organ fusion phenotypes. This study uncovers a role for BR in the spatiotemporal control of organ boundary formation and morphogenesis in the SAM.

Fig. 1.

BRs affect plant architecture. (A–D) BR mutants show altered stem bending. A WT plant (A) has straight main stem, the stem of the bzr1-1D mutant (B) bends toward the axillary branches and cauline leaves, and that of the det2-1 mutant (C) bends away from the lateral branch. (D) Measurement of stem bending toward (positive angle) or away from (negative angle) the lateral branch in WT and various BR mutants. (E–G) SEM image of the junction between stem (s), branch (b), and cauline leaf (c) in WT (E), bzr1-1D (F), and det2-1 (G) plants. (H and I) Light and SEM images of straight silique of WT (H) and bending silique of bzr1-1D (I) with a stamen fused to it (arrow). (J) SEM image showing fusion between a stamen and carpel in a mature flower of bzr1-1D. Lower panel is enlarged portion of upper panel. (K and L) SEM images of stamens in WT (K) and bzr1-1D (L). Arrows show the fused region between stamens in bzr1-1D. (M and N) All WT seedlings (M) have two separate cotyledons, and a small percentage of bzr1-1D seedlings (N) have fused cotyledons. (O and P) SEM images of siliques from WT (O) and bri1-5 (P). Green lines mark the replums (boundaries between valves).

Fig. 2.

BR inhibits organ boundary formation through BZR1-mediated repression of CUC and LOF genes. (A) Quantitative RT-PCR analysis of expression levels of boundary identity genes (CUC1, CUC2, CUC3, LOF1, and LOB) in the first and second axillary branch junctions of WT (Col), bzr1-1D and det2 mutants, and a bzr1-1D-CFP transgenic line with a stronger phenotype than bzr1-1D. (B) The CUC genes are down-regulated by BL. Promoter-GUS transgenic plants were grown on medium containing 10 nM epi-brassinolide (+BL) or 1 μM brassinazole (BRZ) for 9 d and stained for 6 h. (C) LOF1::GUS seedling were grown on 1/2 MS medium with indicated concentration of BL for 10 d in the light before GUS staining. (D) LOB::GUS expression in WT and bzr1-1D. (E) CUC3::GUS expression in the leaf junctions of 2-wk-old seedlings (Top), lateral branch junction (Middle), and inflorescence (Bottom) of WT, bzr1-1D, and bzr1-1D-CFP transgenic line. (F) ChIP-qPCR analysis of BZR1 binding to the promoter regions of CUC1, CUC2, and CUC3 (see promoter maps in Fig. S2). The known BZR1 target gene DWF4 and nontarget CNX5 are used as positive and negative controls. (G and H) bzr1-1D has similar stamen-pistil (G) and stamen-stamen (H) fusion phenotypes to the cuc2;cuc3 double mutant. For each panel from left to right: WT, bzr1-1D, and cuc2;cuc3. (I) Quantitative RT-PCR analysis of the expression levels of LOF1 in WT, cuc2, cuc3, and cuc2;cuc3 mutants grown on 1/2 MS medium for 5 d. All data of relative expression were measured as the ratio to the UBC30 gene. Error bars are SEs.

Fig. 3.

Differential accumulation of BZR1 in the nucleus of primordium and boundary cells is required for proper boundary formation. BZR1-YFP transgenic line of Arabidopsis was crossed with a bzr1-1D-CFP line, and the distributions of BZR1-YFP (A and E) and bzr1-1D-CFP (B and F) in a floral bud (A–C) or apical meristem (E–G) of the F₁ plants were analyzed by confocal microscopy. C and G are merges of panels A with B and E with F. (D and G) SEM images of a floral bud (D) or apical meristem (G) show boundary areas (B) between the sepal primordium (SP) and floral meristem (FM) (A–D) or between shoot apical meristem (SAM) and floral primordium (FP) (E–H).