Supraoptimal Brassinosteroid Levels Inhibit Root Growth by Reducing Root Meristem and Cell Elongation in Rice

Abstract

Root growth depends on cell proliferation and cell elongation at the root meristem, which are controlled by plant hormones and nutrient availability. As a foraging strategy, rice (Oryza sativa L.) grows longer roots when nitrogen (N) is scarce. However, how the plant steroid hormone brassinosteroid (BR) regulates rice root meristem development and responses to N deficiency remains unclear. Here, we show that BR has a negative effect on meristem size and a dose-dependent effect on cell elongation in roots of rice seedlings treated with exogenous BR (24-epicastasterone, ECS) and the BR biosynthesis inhibitor propiconazole (PPZ). A genome-wide transcriptome analysis identified 4110 and 3076 differentially expressed genes in response to ECS and PPZ treatments, respectively. The gene ontology (GO) analysis shows that terms related to cell proliferation and cell elongation were enriched among the ECS-repressed genes. Furthermore, microscopic analysis of ECS- and PPZ-treated roots grown under N-sufficient and N-deficient conditions demonstrates that exogenous BR or PPZ application could not enhance N deficiency-mediated root elongation promotion as the treatments could not promote root meristem size and cell elongation simultaneously. Our study demonstrates that optimal levels of BR in the rice root meristem are crucial for optimal root growth and the foraging response to N deficiency.

Keywords: brassinosteroid; nitrogen deficiency; propiconazole; rice; root meristem.

Figure 1

Effect of ECS and PPZ treatments on rice root growth. Root phenotypes of rice seedlings grown for 5 d under different concentrations of ECS or PPZ or combination of ECS and PPZ (4 μM). (a) Representative images of roots grown under different treatments. Scale bar = 2 cm. (b,c) Quantification of primary root length. Data are means ± SD (n = 10 biological replicates). Significant differences between the treatment and the mock control are indicated by ** for p < 0.001. Significant differences between PPZ and no PPZ (with the same ECS concentration) are indicated by ⁺ and ⁺⁺ for p < 0.05 and 0.001, respectively.

Figure 2

Effect of ECS and PPZ treatments on rice root meristem and cell elongation in primary root tips. Seedlings were grown in the absence and presence of PPZ for 5 d, and then treated with ECS for 24 h. (a) Confocal microscopy images of rice root meristems treated with mock, PPZ (4 μM) or ECS (10 nM). Scale bar = 100 μm. Arrowheads mark the end of the meristem zone; the PPZ-treated root had large meristem that the end of the meristem zone was not present in the image. (b–d) Quantifications of root meristem size (b), meristem cell number (c) and average meristem cell length (d) were determined from cortical cells in the 4th cortical layer by measuring from the QC to the first elongated cell. Mature cell length (e) was determined from the average length of five adjacent mature cortical cells. Data are means ± SD (n ≥ 6 biological replicates). Significant differences between the treatment and the mock control are indicated by * and ** for p < 0.05 and 0.001, respectively. Significant differences between PPZ and no PPZ (with the same ECS concentration) are indicated by ⁺ and ⁺⁺ for p < 0.05 and 0.001, respectively.

Figure 3

Transcriptomic analysis of differentially expressed genes in ECS- and PPZ-treated rice roots. (a) Venn diagram showing the overlap between the lists of significant ECS-induced, ECS-repressed, PPZ-induced and PPZ-repressed genes (|fold change| > 1.5; adjusted p-value < 0.05). The numbers of DEGs are shown in parentheses. (b) Hierarchically clustered heatmap displaying the log₂FC values of all significant genes in the ECS vs. mock or PPZ vs. mock comparisons. (c) GO biological process term enrichment analysis of the ECS and PPZ DEG lists.

Figure 4

Expression of genes involved in BR biosynthesis and signaling, ethylene biosynthesis, cell proliferation and cell elongation. Heatmaps represent log2FC values of genes in the ECS vs. mock or PPZ vs. mock comparisons. Black dots indicate statistical significance of differential expression (adjusted p-value < 0.05). (a) BR biosynthetic and signaling genes, (b) OsPLT genes, (c) ethylene biosynthesis genes, (d,e) cell wall loosening and remodeling genes OsXTHs (d) and OsEXPs (e), (f,g) aquaporin genes OsPIPs (f) and OsTIPs (g). Only genes that showed statistical significance in at least one of the ECS or PPZ comparisons were included in this figure.

Figure 5

Effect of ECS and PPZ treatments on root growth responses to N deficiency. Germinated seeds were grown in normal N for 5 d and then transferred to either normal N or low N conditions containing 10 nM ECS or 4 μM PPZ or mock for 7 d. (a) Representative images of roots grown under different treatments. Scale bar = 2 cm. (b) Crown root length was calculated from the average of the three longest crown roots. Data are means ± SD (n = 10 biological replicates). (c–g) Quantifications of root meristem size (c), meristem cell number (d) and average meristem cell length (e) in the crown roots were determined from cortical cells in the 4th cortical layer by measuring from the QC to the first elongated cell. Mature cell length (f) was determined from the average length of five adjacent mature cortical cells. (g) Average cortical cell length along the longitudinal root axis from the QC illustrated the number of cells in the meristem, the onset of rapid cell elongation and the effect of low N on promoting cell elongation under mock and ECS treatments but not PPZ treatment. Data are means ± SD (n ≥ 6 biological replicates). Significant differences are indicated by * for p < 0.05.