Construction of an Overexpression Library for Chinese Cabbage Orphan Genes in Arabidopsis and Functional Analysis of BOLTING RESISTANCE 4-Mediated Flowering Delay

Abstract

Orphan genes (OGs), which are unique to a specific taxon and have no detectable sequence homology to any known genes across other species, play a pivotal role in governing species-specific phenotypic traits and adaptive evolution. In this study, 20 OGs of Chinese cabbage (Brassica rapa OGs, BrOGs) were transferred into Arabidopsis thaliana by genetic transformation to construct an overexpression library in which 50% of the transgenic lines had a delayed flowering phenotype, 15% had an early flowering phenotype, and 35% showed no difference in flowering time compared to control plants. There were many other phenotypes attached to these transgenic lines, such as leaf color, number of rosette leaves, and silique length. To understand the impact of BrOGs on delayed flowering, BrOG142OE, which showed the most significantly delayed flowering phenotype, was chosen for further analysis, and BrOG142 was renamed BOLTING RESISTANCE 4 (BR4). In BR4OE, the expression of key flowering genes, including AtFT and AtSOC1, significantly decreased, and AtFLC and AtFRI expression increased. GUS staining revealed BR4 promoter activity mainly in the roots, flower buds and leaves. qRT-PCR showed that BR4 primarily functions in the flowers, flower buds, and leaves of Chinese cabbage. BR4 is a protein localized in the nucleus, cytoplasm, and cell membrane. The accelerated flowering time phenotype of BR4OE was observed under gibberellin and vernalization treatments, indicating that BR4 regulates flowering time in response to these treatments. These results provide a foundation for elucidating the mechanism by which OGs regulate delayed flowering and have significance for the further screening of bolting-resistant Chinese cabbage varieties.

Keywords: Arabidopsis; Chinese cabbage; delayed flowering; orphan genes; overexpression library.

Figure 1

Summary figure of the phenotypic investigation of Arabidopsis transgenic plants. The blue bar chart in the figure represents the aggregate results of the multiple phenotypes of different transgenic lines. Purple circles represent lines that bloom late and have other phenotypes. Blue circles represent lines that bloom earlier and have other phenotypes. Yellow circles represent lines with no difference in flowering time but with other phenotypes. Gray circles represent transgenic plants that do not have the corresponding phenotype. EB represents early bolting; BND represents no difference in bolting; DB represents delayed bolting; NRD represents a decreased number of rosette leaves; NRND represents no difference in the number of rosette leaves; NRI represents an increased number of rosette leaves; SHD represents a decrease in stem height; SHND represents no difference in stem height; SHI represents an increase in stem height; RRD represents a decrease in the rosette radius; RRND represents no difference in the rosette radius; RRI represents an increase in the rosette radius; SBD represents a decrease in the number of main stem branches; SBND represents no difference in the number of main stem branches; SBI represents an increase in the number of main stem branches; BSD represents a decrease in the number of main branch siliques; BSND represents no difference in the number of main branch siliques. BSI represents the increase in the number of main branch siliques; SLD represents a decrease in the silique length; SLND represents no difference in the silique length; SLI represents an increase in the silique length. These phenotypes were in comparison to WT.

Figure 2

Phenotypes of transgenic Arabidopsis plants. (a) Flowering stage comparison among BrOG148OE, BrOG140OE, and WT (bar = 10 mm). (b) Phenotype comparison between BrOG144OE and WT (bar = 10 mm). (c) Mature silique of BrOG136OE (bar = 1 mm). (d) Leaf color comparison between BrOG146OE and WT (bar = 10 mm). (e) Leaf color comparison between BrOG129OE and WT (bar = 10 mm). (f) Leaf shape comparison between BrOG135OE and WT (bar = 10 mm). (g) Floral organ comparison among BrOG140OE, BrOG135OE, BrOG144OE, and WT (bar = 1 mm).

Figure 3

Phenotypic characterization of BR4OE lines. (a) Phenotypes of WT and BR4OE plants under short day (SD) (left)/long day (LD) conditions (right) (bar = 10 mm). (b) Floral organ phenotypes of WT and BR4OE (bar = 1 mm). (c) Leaf phenotypes of WT and BR4OE (Bar = 10 mm). (d) Bolting time, flowering time, and rosette leaf number of WT and BR4OE under SD/LD conditions. Experiments were repeated three times (n ≥ 10), and asterisks (***) indicate a significant difference (p < 0.001) from WT, as shown by a Student’s t-test.

Figure 4

Expression of flowering-related genes. All data are shown as the mean ± SD of three biological replicates. Asterisks indicate a significant difference from WT, as shown by a Student’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

Analysis of the expression characteristics of BR4. (a) GUS staining of the BR4 promoter (bar = 1 mm). (b) Expression levels of BR4 in various tissues during the flowering stage. All data are shown as the mean ± SD of three biological replicates. Asterisks indicate a significant difference from WT, as shown by a Student’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

Localization of the BR4 protein in tobacco leaf cells. A Leica confocal microscope was used to collect images 48 h after agro-infiltration. Bar = 75 μm.

Figure 7

Flowering time of BR4OE and WT plants under different treatments. (a,b) Phenotypic observation of WT and BR4OE under GA₃ treatment. (c,d) Phenotypic observation of WT and BR4OE under vernalization treatment. All data are shown as the mean ± SD of three biological replicates. Asterisks indicate a significant difference from WT, as shown by a Student’s t-test; ns indicates no difference, ^ns p > 0.05, * p < 0.05, *** p < 0.001. Ver represents vernalization treatment.

Figure 8

BR4 expression levels in Chinese cabbage under different treatments: (a) GA₃ treatment, (b) vernalization treatment. Ver represents vernalization treatment.