Functional characterization of GmBZL2 (AtBZR1 like gene) reveals the conserved BR signaling regulation in Glycine max

Abstract

Brassinosteroids (BRs) play key roles in plant growth and development, and regulate various agricultural traits. Enhanced BR signaling leads to increased seed number and yield in Arabidopsis bzr1-1D (AtBZR1(P234L), gain-of-function mutant of the important transcription factor in BR signaling/effects). BR signal transduction pathway is well elucidated in Arabidopsis but less known in other species. Soybean is an important dicot crop producing edible oil and protein. Phylogenetic analysis reveals AtBZR1-like genes are highly conserved in angiosperm and there are 4 orthologues in soybean (GmBZL1-4). We here report the functional characterization of GmBZL2 (relatively highly expresses in flowers). The P234 site in AtBZR1 is conserved in GmBZL2 (P216) and mutation of GmBZL2(P216L) leads to GmBZL2 accumulation. GmBZL2(P216L) (GmBZL2*) in Arabidopsis results in enhanced BR signaling; including increased seed number per silique. GmBZL2* partially rescued the defects of bri1-5, further demonstrating the conserved function of GmBZL2 with AtBZR1. BR treatment promotes the accumulation, nuclear localization and dephosphorylation/phosphorylation ratio of GmBZL2, revealing that GmBZL2 activity is regulated conservatively by BR signaling. Our studies not only indicate the conserved regulatory mechanism of GmBZL2 and BR signaling pathway in soybean, but also suggest the potential application of GmBZL2 in soybean seed yield.

Figure 1. Phylogenetic relationships and exon-intron structures of BZR1-like genes.

(a) Phylogenetic tree of BZR1-like genes. The bootstrap values (>50%) of Maximum likelihood and Neighbor-Joining analyses are shown next to the nodes. (b) Schematic representation of exon-intron structures. Exons and introns are represented by boxes and lines, respectively. The N-terminal domain and PEST domain are indicated with red and blue boxes, respectively. The conserved proline site (P234L mutated in bzr1-1D) in the PEST domain is represented by an orange matchstick, and the putative conserved proline site that is homologous to that in PEST domain is indicated with a gray matchstick.

Figure 2. The structures of GmBZL2 and tissue specific expression pattern in Arabidopsis.

(a) A schematic diagram of GmBZL2 with two exons and an intron, which are represented with black boxes and intervening line. The start codon ATG starts from the +1 nucleotide, and the stop codon TAG stops at +1931 nucleotide. (b) The conserved domain of GmBZL2. There are two domains in the GmBZL2 sequence. BES1-N domain including 130 amino acids shares high similarity with N-terminal of AtBES1. The PEST domain contains the deduced conserved proline site P216 similar to that of AtBZR1. (c) The multiple alignment of the amino acid sequences in the PEST domain of bzr1-1D, BZR1, GmBZL2. The conserved proline residue was shown in red rectangle. (d) The GUS staining of 7-day-old seedling in GmBZL2-GUS transgenic line, bar = 2 mm; (e) rosette leaf, bar = 2 mm; (f) inflorescence, bar = 1 mm; (g) open flower, bar = 0.5 mm; (h–k) pistils from early stage to late stage, bars = 0.5 mm; (l) the silique after fertilization, bar = 0.5 mm.

Figure 3. GmBZL2 has the conservative function in BR signaling pathway in Arabidopsis.

(a,b) The tobacco epidermal cell expressing 35S::GmBZL2-GFP without BL treatment and with 8 hr BL treatment, bars = 50 μm. (c) The dephosphorylated GmBZL2 increased after 10 hr BL treatment with Western blot detection, ribosome bands were used as loading control. The green and red arrows indicate the phosphorylated and dephosphorylated GmBZL2. The five protein marker belts are 170 kD, 130 kD, 100 kD, 70 kD and 55 kD from top to bottom, LC indicates loading control. (d) Confocal images of cotyledon epidermal cells from 7-day-old seedling of GmBZL2-GFP and GmBZL2*-GFP transgenic lines. The blue and green channels correspond to the nuclear-associated DAPI fluorescence and GmBZL2-GFP (GmBZL2*-GFP) fluorescence, respectively. Overlay shows both channels in images. Bars = 50 μm. (e) The ratio of dephosphorylated/phosphorylated GmBZL2 is higher in GmBZL2*-GFP than in GmBZL2-GFP lines, ribosome bands were used as loading control. The green and red arrows indicate the phosphorylated and dephosphorylated proteins, respectively. The five protein marker belts are 170 kD, 130 kD, 100 kD, 70 kD and 55 kD from top to bottom, LC indicates loading control.

Figure 4. The characterization of the GmBZL2*-GUS transgenic lines.

(a) Four-week-old plants of the Col-0, GmBZL2*-GUS transgenic lines. Bar = 2.5 cm. (b) The rosette leaves from the plants of Col-0 and GmBZL2*-GUS transgenic lines from the top to the bottom. Bar = 2 cm. (c) Six-week-old plants of the Col-0, bzr1-1D, and GmBZL2*-GUS transgenic lines. Bar = 2 cm. (d) The transparent siliques of Col-0 and GmBZL2*-GUS transgenic lines. Bar = 4 mm. (e) Statistical analysis of seed number per silique from the 3^rd to 13^th silique on the primary stem (a total of 5 plants from each line) of wild type and transgenic lines. The student t test was used to analyze the significant differences between wild type and GmBZL2*-GUS transgenic lines (*p < 0.05, **p < 0.01). (f) The 6-day-old seedlings of Col-0 without BRZ treatment, Col-0, bzr1-1D, and GmBZL2*-GUS transgenic lines grown on the 1/2MS medium with BRZ in dark. Bar = 1 cm. (g) Statistical analysis of the length of 6-day-old seedling hypocotyls of wild type. bzr1-1D and GmBZL2*-GUS transgenic lines with BRZ treatment in dark condition (a total of 15 plants from each line). The student t test was used to analyze the significant differences between the wild type and bzr1-1D, GmBZL2*-GUS transgenic lines with BRZ treatment (**p < 0.01). (h) The expression level of CPD and DWF4 in 10-day-old seedlings of Col-0, bzr1-1D, and GmBZL2*-GUS transgenic lines.

Figure 5. The characterization of the GmBZL2*-GFP transgenic lines.

(a) The 4-week-old plants of bzr1-1D, Col-0 and the GmBZL2*-GFP lines. Bar = 2.5 cm. (b) The rosette leaves from the plants of bzr1-1D, Col-0 and GmBZL2*-GFP transgenic lines from the top to the bottom. Bar = 2 cm. (c) The siliques of GmBZL2*-GFP transgenic lines and Col-0. Bar = 1 cm. (d) The 7-week-old seedlings of Col-0, bzr1-1D and the GmBZL2*-GFP transgenic lines. Bar = 5 cm. The kink inflorescence stem in the red box is the typical bzr1-1D like phenotype. (e) The transparent siliques of Col-0, bzr1-1D, and GmBZL2*-GFP trangenic lines. Bar = 4 mm. (f) Statistical analysis of seed number per silique from the 3^rd to 13^th silique on the primary stem (a total of 5 plants from each line) of wild type, bzr1-1D and transgenic lines. The student t test was used to analyze the significant differences among wild type, bzr1-1D, and independent GmBZL2*-GFP transgenic lines (***p < 0.001). (g) The 6-day-old seedling of Col-0 without BRZ treatment; the Col-0 and GmBZL2*-GFP transgenic lines grow on the 1/2MS medium with BRZ in dark. Bar = 1 cm. (h) Statistical analysis of 6-day-old seedling hypocotyl length of wild type (with and without BRZ treatment), bzr1-1D and GmBZL2*-GFP transgenic lines with BRZ treatment in dark (a total of 15 plants from each line). The student t test was used to analyze the significant differences between wild type and bzr1-1D, GmBZL2*-GFP transgenic lines with BRZ treatment (***p < 0.001). (i) The expression level of CPD and DWF4 in 10-day-old seedlings of Col-0, bzr1-1D, and GmBZL2*-GFP transgenic lines.

Figure 6. GmBZL2* partially rescues the BR-deficient phenotype of bri1-5.

(a) The overview of 6-week-old plants of bri1-5, GmBZL2-GUS/bri1-5 and GmBZL2*-GUS/bri1-5 transgenic lines. Bar = 3 cm. (b) The petioles of bri1-5, wild type (WS) and GmBZL2*-GUS/bri1-5 transgenic lines. Bar = 1 cm. (c) The siliques of bri1-5 and the GmBZL2*-GUS/bri1-5 lines. Bar = 0.5 cm. The student t test was performed between bri1-5 and the transgenic lines (***P < 0.001). (d) Seed number per silique of WS, bri1-5 and GmBZL2*-GUS/bri1-5 transgenic lines. Bar = 0.5 cm. (e) Statistical analysis of seed number from the 3^rd to 13^th silique on the primary stem (a total of 5 plants from each line) of wild type, bri1-5 and transgenic lines. The student t test was used to analyze the significant differences between bri1-5 and GmBZL2*-GUS/bri1-5 transgenic lines (***p < 0.001). (f) The 6-day-old seedling of WS, bri1-5 and GmBZL2*-GUS/bri1-5 transgenic lines grown in dark condition. Bar = 1 cm. (g) Statistical analysis of the 6-day-old seedling hypocotyl length of WS, bri1-5 and GmBZL2*-GUS/bri1-5 transgenic lines in dark (a total of 15 plants from each line). The student t test was used to analyze the significant differences between bri1-5 and GmBZL2*-GUS/bri1-5 transgenic lines (***p < 0.001).