Evolutionary analysis and functional characterization of SiBRI1 as a Brassinosteroid receptor gene in foxtail millet

Abstract

Brassinosteroids (BRs) play important roles in plant growth and development. Although BR receptors have been intensively studied in Arabidopsis, those in foxtail millet remain largely unknown. Here, we show that the BR signaling function of BRASSINOSTEROID INSENSITIVE 1 (BRI1) is conserved between Arabidopsis and foxtail millet, a new model species for C4 and Panicoideae grasses. We identified four putative BR receptor genes in the foxtail millet genome: SiBRI1, SiBRI1-LIKE RECEPTOR KINASE 1 (SiBRL1), SiBRL2 and SiBRL3. Phylogenetic analysis was used to classify the BR receptors in dicots and monocots into three branches. Analysis of their expression patterns by quantitative real-time PCR (qRT-PCR) showed that these receptors were ubiquitously expressed in leaves, stems, dark-grown seedlings, roots and non-flowering spikelets. GFP fusion experiments verified that SiBRI1 localized to the cell membrane. We also explored the SiBRI1 function in Arabidopsis through complementation experiments. Ectopic overexpression of SiBRI1 in an Arabidopsis BR receptor loss-of-function mutant, bri1-116, mostly reversed the developmental defects of the mutant. When SiBRI1 was overexpressed in foxtail millet, the plants showed a drooping leaf phenotype and root development inhibition, lateral root initiation inhibition, and the expression of BR synthesis genes was inhibited. We further identified BRI1-interacting proteins by immunoprecipitation (IP)-mass spectrometry (MS). Our results not only demonstrate that SiBRI1 plays a conserved role in BR signaling in foxtail millet but also provide insight into the molecular mechanism of SiBRI1.

Keywords: BRI1; Brassinosteroids; Foxtail millet; Phylogenetic analysis.

Fig. 1

Phylogenetic evolutionary tree and gene structures of BRI1 gene family members. (A), an neighbor-joining (NJ) phylogenetic tree was constructed using the full-length protein sequence alignments of BRI1 genes identified using MUSCLE in MEGAX. Bootstrap supports were indicated by the colour of the branch. The OTUs are labelled as follows: Dicotyledons (blue); Monocotyledons (red); Amborella trichopoda (black). The colour blocks indicate the types, with type I (pink), type II (green), and type III (yellow) denoted. (B), Gene structures of the BRI or BRL genes. The lengths of rectangles and lines are scaled according to the mRNA lengths. CDSs (green rectangles), UTRs (yellow rectangles), and introns (black line) are denoted

Fig. 2

Expression and subcellular localization analysis of SiBRI1 and its orthologues in different tissues in foxtail millet. (A), Leaves (L), stems (S), dark-grown seedlings (DGS), roots (R), non-flowering spikelets (NFS), and dry seeds (DS). Error bars indicate the mean ± standard deviation (SD). N = 3. Statistically significant differences are indicated by different lowercase letters (p < 0.05, one-way ANOVA with Tukey’s significant difference test). (B), Confocal images indicate the localization of SiBRI1-eGFP in the roots of 3-day-old dark-grown seedlings overexpressing SiBRI1 with a GFP tag at the C-terminus. Scale bar = 20 µm

Fig. 3

SiBRI1 overexpression rescued the mutant phenotypes of bri1-116 plants. (A), Phenotypes of light-grown 8-week-old Col-0 and bri1-116 mutant plants overexpressing SiBRI1 with a C-terminal YFP tag. An enlarged view of bri1-116 is shown in the white box. Scale bar = 1 cm. (B), Expression levels of SiBRI1-YFP and AtBZR1 in the transgenic plants shown in (A). The differential accumulation pattern of AtBZR1 in (A) was detected by anti-BZR1; pAtBZR1 showed the phosphorylation form of AtBZR1, and AtBZR1 showed the unphosphorylated form of AtBZR1. Anti-HSP70 and Ponceau S staining of the Rubisco large subunit was used as an equal loading control. The bottom gel shows the genotyping identification of the transgenic plants in (A)

Fig. 4

SiBRI1 overexpression activated BR signaling in bri1-116 plants. (A), Phenotypes of Col-0 and bri1-116 mutant plants overexpressing SiBRI1 that were grown in the presence of the indicated concentration of BL for 7 days. Scale bar = 1 cm. (B), Relative root lengths of the plants in (A). Error bars indicate the mean ± standard deviation (SD). N > 30. Statistically significant differences are indicated by different lowercase letters (p < 0.05, two-way ANOVA with Tukey’s significant difference test). (C), Phenotypes of Col-0 and bri1-116 mutant plants overexpressing SiBRI1 that were grown in the presence of the indicated concentration of PCZ for 7 days. Scale bar = 1 cm. (D), Relative hypocotyl lengths of the plants in (C). Error bars indicate the mean ± standard deviation (SD). N > 30. Statistically significant differences are indicated by different lowercase letters (p < 0.05, two-way ANOVA with Tukey’s significant difference test). (E), Immunoblot analysis of BZR1 in transgenic plants overexpressing SiBRI1 in the bri1-116 mutant or wild type background under PCZ. pAtBZR1 showed the phosphorylation form of AtBZR1, and AtBZR1 showed the unphosphorylated form of AtBZR1. Expression levels of HSP70 and AtBZR1 in the transgenic plants shown in (C). Ponceau S staining of the Rubisco large subunit and the expression level of HSP70 was used as an equal loading control

Fig. 5

SiBRI1 regulated the BR response in foxtail millet. (A), Phenotype of light-grown 4-leaf stage Ci846 plants and two independent lines of pUbi:SiBRI1-eGFP/Ci846 (SiBRI1-OX); scale bar = 1.5 cm. (B), Expression levels of SiBRI1-YFP in the transgenic plants shown in (A). Ponceau S staining of the Rubisco large subunit was used as an equal loading control. (C), Quantitative real-time RT-PCR analysis of SiCPD, SiD2, SiDWARF and SiDWF4 expression in the roots of 8-day-old seedlings overexpressing SiBRI1 under 1 μM BL immersion for 1 h. Three biological repetitions were established. Error bars indicate the mean ± standard deviation (SD). Statistically significant differences are indicated by different lowercase letters (p < 0.05, one-way ANOVA with Tukey’s significant difference test)

Fig. 6

SiBRI1-OX was hypersensitive to BL in foxtail millet. (A), Phenotypes of Ci846 plants overexpressing SiBRI1 that were grown in the presence of the indicated concentration of BL for 6 days under 16L/8D 28 °C. Bar = 1 cm. (B), Relative root lengths of the plants in (A). N > 12. Error bars indicate the mean ± standard deviation (SD). Statistically significant differences are indicated by different lowercase letters (p < 0.05, two-way ANOVA with Tukey’s significant difference test). (C), The lateral root numbers of seedlings in (A). N > 12. Lateral roots with lengths greater than 1 mm are marked as elongated (E), those with lengths less than 2 mm are marked as unelongated (NE)

Fig. 7

Quantitative RT-PCR analysis of SiPLT-L1 and SiLBD16 expression in the roots of 8-day-old SiBRI1 overexpressing plants. The transgenic plants and Ci846’s roots were immersed under 1 μM BL for 1 h. Two–three biological repetitions. Error bars indicate the mean ± standard deviation (SD). Statistically significant differences are indicated by different lowercase letters (p < 0.05, one-way ANOVA with Tukey’s significant difference test)

Fig. 8

SiBRI1 interaction proteins under BL treatment. (A). The Venn diagram of the proteins identified in pUbi:SiBRI1-eGFP/Ci846 under BL treatment. 8-days-old Ci846 and pUbi:SiBRI1-eGFP/Ci846 seedlings was treated with 1 µM BL for 2 h and the overground tissues used to extract the interaction proteins with SiBRI1 by GFP-Trap. (B) GO annotation analysis of the SiBRI1 interaction proteins in pUbi:SiBRI1-eGFP/Ci846 under BL treatment or not, Gene ontology (GO) annotation was performed online using AgriGO (http://bioinfo.cau.edu.cn/agriGO/), the numbers in bar polots represent the fold enrichment compared with the whole-genome level