GmUFO1 Regulates Floral Organ Number and Shape in Soybean

Abstract

The UNUSUAL FLORAL ORGANS (UFO) gene is an essential regulatory factor of class B genes and plays a vital role in the process of inflorescence primordial and flower primordial development. The role of UFO genes in soybean was investigated to better understand the development of floral organs through gene cloning, expression analysis, and gene knockout. There are two copies of UFO genes in soybean and in situ hybridization, which have demonstrated similar expression patterns of the GmUFO1 and GmUFO2 genes in the flower primordium. The phenotypic observation of GmUFO1 knockout mutant lines (Gmufo1) showed an obvious alteration in the floral organ number and shape and mosaic organ formation. By contrast, GmUFO2 knockout mutant lines (Gmufo2) showed no obvious difference in the floral organs. However, the GmUFO1 and GmUFO2 double knockout lines (Gmufo1ufo2) showed more mosaic organs than the Gmufo1 lines, in addition to the alteration in the organ number and shape. Gene expression analysis also showed differences in the expression of major ABC function genes in the knockout lines. Based on the phenotypic and expression analysis, our results suggest the major role of GmUFO1 in the regulation of flower organ formation in soybeans and that GmUFO2 does not have any direct effect but might have an interaction role with GmUFO1 in the regulation of flower development. In conclusion, the present study identified UFO genes in soybean and improved our understanding of floral development, which could be useful for flower designs in hybrid soybean breeding.

Keywords: GmUFOs; floral organ number; floral organ shape; knockout; soybean.

Figure 1

Phylogenetic tree and multiple sequence alignment of UFO genes. (A) Phylogenetic analysis of the selected 11 UFO proteins from nine species, namely Arabidopsis thaliana (AT1G30950), Glycine max (Glyma.05G134000, Glyma.08G088700), Medicago truncatula (Medtr4g094748), Vigna unguiculate (Vigun03g153100), Phaseolus vulgaris (Phvul.002G188300), Gossypium raimondii (Gorai.002G139700), Oryza sativa (Os06g45460), Zea mays (GRMZM2G109966, GRMZM2G360081), and Hordeum vulgare (Hr1G108970). The neighbor-joining tree was constructed using MEGA11, and the bootstrap value was set at 1000 replicates. (B) Amino acid sequence alignment of UFOs in soybean and other species. The orange line represents the F-box domain; the black shade represents the conserved amino acids, and the gray shade represents the relatively conserved amino acids.

Figure 2

The expression profile of GmUFO1. (A) In situ hybridization of the GmUFO1 gene in the soybean flower primordium of different development stages, and the signals were purple. Bar = 100 μm. (B) Scanning electron microscopy (SEM) observations of in situ hybridization and stereoscopic models of GmUFO1 gene expression at five developmental stages. The areas of gene expression were shown in blue and the red lines represented the slice position corresponding to (A). Bar = 100 μm. (C) Expression location of GmUFO1-GFP in Arabidopsis thaliana. The GFP signals were green and mCherry signals were red. Bar = 10 μm.

Figure 3

Summary of CRISPR/Cas9-mediated genome editing of GmUFO genes. (A) Structural diagram of the CRISPR/Cas9 vector. (B) Target sites designed for GmUFO1 and GmUFO2 in CRISPR/Cas9, and the PAM sequence was in red. (C) Summary of mutation frequency in the T1 generation. (D–F) Seedling phenotype of Gmufo1 single mutant lines (D), Gmufo2 single mutant lines (E), and Gmufo1ufo2 double mutant lines (F) compared with the wild type. Scale bar = 10 cm.

Figure 4

Identification of the GmUFO1 knockout lines. (A) Floral organ phenotype of the three Gmufo1 mutant lines compared with the wild type. Scale bar = 1 mm. (B) Statistics of the number of sepals, stamens, and petals in Gmufo1 mutant lines. Significant differences according to two-sided Student’s t test (* p < 0.05, ** p  <  0.01). Data are means ±  SD for at least 10 flowers in each line. (C) ABC function gene expression was analyzed in the flowers of Williams 82, Gmufo1-1, Gmufo1-15, and Gmufo1-16. All data presented are mean ± SD for three biological replicates. Asterisks indicate significant differences according to two-sided Student’s t test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). (D) GmUFO1 and GmUFO2 expression level in three mutant lines and the wild type. (E) CRISPR/Cas9 target site sequencing of GmUFO1 and GmUFO2 in the three selected lines.

Figure 5

Identification of the GmUFO2 knockout lines. (A) Floral organ phenotype of the three Gmufo2 mutant lines compared with the wild type. Scale bar = 1 mm. (B) Statistics of the number of sepals, stamens, and petals in Gmufo2 mutant lines. Non-significant differences were found according to two-sided Student’s t test. Data are means ±  SD for at least 10 flowers of each line. (C) ABC model gene expression was analyzed in the flowers of Williams 82, Gmufo2-1, Gmufo2-2, and Gmufo2-3. All data presented are mean ± SD for three biological replicates. Asterisks indicate significant differences according to two-sided Student’s t test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). (D) GmUFO1 and GmUFO2 expression level in three mutant lines and the wild type. (E) CRISPR/Cas9 target site sequencing of GmUFO1 and GmUFO2 in the three selected lines.

Figure 6

Identification of the GmUFO1 and GmUFO2 knockout lines. (A) Floral organ phenotype of the three Gmufo1ufo2 mutant lines compared with the wild type. Scale bar = 1 mm. (B) Statistics of the number of sepals, stamens, and petals in the Gmufo1ufo2 mutant lines. Non-significant differences were found according to two-sided Student’s t test. Data are means ±  SD for at least 10 flowers in each line. (C) ABC model gene expression was analyzed in the flowers of Williams 82, Gmufo1ufo2-4, Gmufo1ufo2-17, and Gmufo1ufo2-18. All data presented are the means ± SD for three biological replicates. Asterisks indicate significant differences according to two-sided Student’s t test (** p < 0.01, *** p < 0.001, **** p < 0.0001). (D) GmUFO1 and GmUFO2 expression level in three mutant lines and the wild type. (E) CRISPR/Cas9 target site sequencing of GmUFO1 and GmUFO2 in the three selected lines.