Molecular Evaluation of the Effects of FLC Homologs and Coordinating Regulators on the Flowering Responses to Vernalization in Cabbage (Brassica oleracea var. capitata) Genotypes

Abstract

The flowering loci of cabbage must be understood to boost their productivity. In this study, to clarify the flowering mechanisms of cabbage, we examined the three flowering repressors BoFLC1, 2 and 3, and the flowering regulators BoGI, BoCOOLAIR, and BoVIN3 of early (CAB1), middle (CAB3), and late (CAB5) flowering cabbage genotypes. Analysis of allele-specifically amplified genomic DNA and various sequence alignments demonstrated that maximal insertions and deletions influenced cabbage flowering behavior, notably in CAB3 and CAB5. Phylogenetic studies showed that BoFLC1, 2, and 3 in the CAB1, 3, and 5 genotypes had the highest homologies to other Brassica species, with CAB3 and 5 the most similar. Although CAB3 and CAB5 have comparable genetic patterns, flowering repressors and flowering regulators were investigated individually with and without vernalization to determine their minor flowering differences. The expression investigation revealed that vernalized CAB5 downregulated all BoFLC genes compared to CAB3 and, in contrast, CAB3 exhibited upregulated BoCOOLAIR. We hypothesized that the CAB3 BoFLC locus' additional insertions may have led to BoCOOLAIR overexpression and BoFLC downregulation. This study sheds light on cabbage genotypes-particularly those of CAB1 and CAB5-and suggests that structural variations in BoFLC2 and 3 bind flowering regulators, such as COOLAIR, which may affect cabbage flowering time.

Keywords: BoFLC; cabbage flowering; flowering regulator; structural variation; vernalization.

Figure 1

Insertion and deletion profiles of the BoFLC genes including BoFLC1 (A), BoFLC2 (B), and BoFLC3 (C) in the genomic DNA of the three flowering lines CAB1, 3, and 5 along with the structure of the reference gene AY306124.1.

Figure 2

Neighbor-joining tree showing phylogenetic relationships based on decoded BoFLC amino acid sequences among early- (CAB1), mid- (CAB3), and late (CAB5)-flowering cabbages. The GenBank accession number is shown in parentheses, and sequences were compared with the three cabbage genotypes used in our study. Bootstrap values ˃ 50% are shown above the branches. At, A. thaliana; Bc, B. carinata; Bj, B. juncea; Bn, B. napus; Bni, B. nigra; Bo, B. oleracea; Br, B. rapa.

Figure 3

mRNA expression patterns of three BoFLC homologs using cDNA from vernalization (dotted lines) and non-vernalization (solid lines) treatments between mid- (CAB 3) and late (CAB 5)- flowering cabbages. Relative expressions of BoFLC1 (top panels A,B), BoFLC2 (middle panels C,D), and BoFLC3 (bottom panels E,F) are shown for CAB 3 (left panels A,C,E, respectively) and CAB 5 (right panels B,D,F, respectively). Different letters indicate statistically significant differences in gene expression across vernalization according to Duncan’s multiple range test at p ≤ 0.05.

Figure 4

mRNA expression patterns of the flowering regulator genes BoGI (GI, top panel A,B), BoCOOLAIR (middle panel C,D), and BoVIN3 (bottom panel E,F) between mid- (CAB 3) and late (CAB 5)-flowering cabbage lines under vernalization (dotted lines) and non-vernalization (solid lines) treatments. The relative expressions of GI, BoCOOLAIR, and BoVIN3 were compared for the mid-flowering CAB 3 line (left A,C,E) and late-flowering CAB 5 line (right B,D,F). The bars indicate standard errors of the means for each week of the vernalization treatment.

Figure 5

Schematic representation of FLC expression and suppression under control and vernalization conditions along with FLC regulator activity on three different BoFLC genes in CAB3 and CAB5 cabbages (w = weeks; green color arrows = CAB3 FLC expressions; red color arrows = CAB5 FLC expressions; maximum dotted lines = expression of BoFLC2; minimal dotted lines = expression of BoFLC3; solid lines = expression of BoFLC1).