SEUSS Integrates Gibberellin Signaling with Transcriptional Inputs from the SHR-SCR-SCL3 Module to Regulate Middle Cortex Formation in the Arabidopsis Root

Abstract

A decade of studies on middle cortex (MC) formation in the root endodermis of Arabidopsis (Arabidopsis thaliana) have revealed a complex regulatory network that is orchestrated by several GRAS family transcription factors, including SHORT-ROOT (SHR), SCARECROW (SCR), and SCARECROW-LIKE3 (SCL3). However, how their functions are regulated remains obscure. Here we show that mutations in the SEUSS (SEU) gene led to a higher frequency of MC formation. seu mutants had strongly reduced expression of SHR, SCR, and SCL3, suggesting that SEU positively regulates these genes. Our results further indicate that SEU physically associates with upstream regulatory sequences of SHR, SCR, and SCL3; and that SEU has distinct genetic interactions with these genes in the control of MC formation, with SCL3 being epistatic to SEU. Similar to SCL3, SEU was repressed by the phytohormone GA and induced by the GA biosynthesis inhibitor paclobutrazol, suggesting that SEU acts downstream of GA signaling to regulate MC formation. Consistently, we found that SEU mediates the regulation of SCL3 by GA signaling. Together, our study identifies SEU as a new critical player that integrates GA signaling with transcriptional inputs from the SHR-SCR-SCL3 module to regulate MC formation in the Arabidopsis root.

Figure 1.

SEU represses MC formation in the root endodermis. A to F, Occurrence of MC formation in roots of Col-0 (A and D), seu-3 (B and E), and 35S::SEU (C and F) at the indicated time points (4 d or 7 d after sowing). The black outlines and white arrowheads highlight the occurrence of MC formation in the root endodermis. Scale bars = 50 μm. G, Time-course analysis of proportion of plants with MC formation in roots of Col-0 and seu-3 seedlings. Error bars represent sd of three independent experiments (n ≥ 25). Bars with different letters are significantly different at P < 0.05, ANOVA. H, Quantitative analysis of proportion of plants with MC formation in roots of Col-0 and 35S::SEU seedlings 7 d after sowing. Error bars represent sd of three independent experiments (n ≥ 25). **, P < 0.01, Student’s t test.

Figure 2.

SEU physically associates with promoter regions of SHR, SCR, and SCL3 and positively regulates their expression. A to H, Expression of SHR::H2B-YFP (A and B), SCR::H2B-YFP (C and D), SCL3::GUS (E and F), and SCL3::SCL3-GFP (G and H) in root tips of Col-0 (A, C, E, and G) and seu-3 (B, D, F, and H) seedlings. Scale bars = 50 μm. I, Yeast one-hybrid assay. The boundaries of promoter regions of SCR, SHR, and SCL3 used for reporter constructs are shown. Yeast cells cotransformed with each combination of reporter and SEU-GAL4-AD constructs (+) were tested for β-galactosidase assay (blue color). Yeast cells transformed only with each of the reporter constructs were used as controls (−). J to L, ChIP-qPCR assay. The relative ChIP recovery of each region in the SEU::SEU-GFP line was expressed as percentage of input DNA (y axis). Error bars represent sd of three independent experiments. Bars with different letters are significantly different at P < 0.05, ANOVA. nA, No antibody; Ab, GFP antibody.

Figure 3.

SEU has distinct genetic interactions with SHR, SCR, and SCL3. A to I, Occurrence of MC formation in roots of Col-0 (A), seu-3 (B), seu-4 (C), shr-2 (D), scr-3 (E), scl3 (F), seu-4 shr-2 (G), seu-3 scr-3 (H), and seu-3 scl3 (I) 7 d after sowing. The black outlines and white arrowheads highlight the occurrence of MC formation in the root endodermis. Scale bars = 50 μm. J and K, Expression of CYCD6;1-GFP-GUS in roots of wild-type (WT; J) and seu-3 (K) seedlings 5 d after sowing. Scale bar = 50 μm. L and M, Quantitative analysis of proportion of plants with MC formation in roots of Col-0, seu-3, seu-3 scr-3, and seu-3 scl3 seedlings 7 d after sowing. Error bars represent sd of three independent experiments (n ≥ 15). Bars with different letters are significantly different at P < 0.05, ANOVA.

Figure 4.

SEU mediates the regulation of SCL3 by GA signaling. A to C, Expression of SEU::GUS (A) and SCL3::GUS (B and C) in root tips of Col-0 (A and B) and seu-3 (C) seedlings treated with mock, 10 μm GA₃, or 10 μm PAC. Scale bars = 50 μm. D, qRT-PCR analysis of SEU and SCL3 transcript levels in roots of Col-0, ga1, and seu-3 seedlings treated with mock, 10 μm GA₃, or 10 μm PAC. Transcript levels from the mock were set to 1. GAPC (AT3G04120) was chosen as the reference gene. Error bars represent sd of three biological replicates. Bars with different letters are significantly different at P < 0.05, ANOVA.

Figure 5.

A simplified yet complex regulatory network for MC formation. In the Arabidopsis root meristem, MC forms by periclinal cell division in the endodermis in a developmental stage-dependent manner. Previous studies (black arrows and inhibition signs) have revealed that GA and several GRAS family transcription factors, including SHR, SCR, SCL3, and DELLAs, are involved in the precise control of MC formation. SHR (at high abundance, which inhibits transcription of its direct target CYCD6;1), SCR, and their direct target SCL3 (which represses its own transcription) inhibit MC formation, whereas SHR (at low abundance, which induces transcription of CYCD6;1) and DELLAs (which are direct upstream regulators of SCL3 and known to induce the expression of early GA biosynthesis genes) promote it. GA triggers degradation of DELLAs and may influence abundance of SHR, thus preventing MC formation. In this study (green arrow and inhibition sign or red arrows), we show that SEU is a critical new player that integrates GA signaling with the transcription inputs from the SHR-SCR-SCL3 module to regulate MC formation. SEU is transcriptionally repressed by GA. SEU is a transcriptional activator of SHR, SCR, SCL3, and DELLAs, and physically associates with promoter regions of SCR, SHR, and SCL3 (red arrows). The colors orange and blue indicate factors that positively (orange) or negatively (blue) regulate MC formation, respectively. EN, Endodermis.