Structural analysis of BBX proteins
The sequence of the first B-box (B1) motif contained in these genes were C-X2-C-X4-A-X3-C-X2-D-X4-C-X2-C-D (Fig. 1A), and the sequence of the second B-box (B2) motif were C-X7-C-X2-C-D-X3-H (Fig. 1B). The distance between the two B-box domains was 5–20 amino acids. It has been found that the conserved amino acid residues in the B-box motifs are very important in regulating protein–protein interaction and transcriptional regulation. According to the reported article, the second B-box motif of AtBBX21 in Arabidopsis was key to bind to HY5 promoter and promote the transcription of HY5. The sequence of CCT domain is R-X5-R-Y-X-E-K-X3-R-X3-K-X2- R-Y-X2-R-K-X2-A-X2-R-X-R-X-K-G-R-F-X-K (Fig. 1C).
In order to understand more clearly the relationship between these genes, a phylogenic tree was constructed according to BBX genes obtained from Pyrus bretschneideri, O. sativa, A. thaliana, P. trichocarpa, Z. mays and Gossypium hirsutum. These BBX proteins were divided to five structural groups (Fig. 2). The structural group I contains two B-box domains and one CCT domain. The structural group II contains also two B-box domains and one CCT domain, but there are some differences in the second B-box domain between the group I and group II. The structural group III consists of a B-box domain and a CCT domain. The structural group IV contains two B-box domains without CCT structure. The structural group V contains only one B-box domain. In cotton, there are only 24 GhBBX genes with CCT structure, accounting for 38% of all GhBBX genes. Among them, 11 GhBBX genes belong to the group I, and 7 GhBBX genes are in the group II and 6 GhBBX genes are in the group III. The remaining are shared into the groups IV and V containing 31 and 8 GhBBX genes, respectively (Fig. 2). Nonetheless, the GhBBX16 is found to fit in the structural group II, which only contains a B-box (Fig. 2). These results indicate that some of the GhBBX proteins can lose a domain in recent evolutionary events, but retain other common features of their structural groups. Phylogenetic analysis shows that the GhBBX proteins belonging to the same structural group are classified by amino acid similarity and the structural organization of B-box and CCT domains.
Structural analysis of BBX genes in cotton
The gene structures of these BBX genes were analyzed in upland cotton. The results indicated that only four BBX genes had no introns, all of which belong to the structural group IV (Fig. 3). The remaining BBX genes contained 1 to 9 introns, but GhBBX40 had the most introns (Fig. 3). GhBBX4, GhBBX9, GhBBX12, GhBBX13, GhBBX18 contained four introns and the same numbers of exons. In addition, there were 26 BBX genes with two introns and one exon, and there were 11 BBX genes with three exons and the same numbers of introns, which belong to the structural group IV and V without CCT (Fig. 3).
Expression pattern of partial BBX family genes in cotton
In order to understand the gene expression pattern of the BBX family genes, qRT-PCR were carried out. The expression levels of these BBX genes from the structural group IV and structural group V were tested at 6 days post anthesis (DPA), 12 DPA, 18 DPA and 24 DPA in brown cotton fibers (Fig. 4). The experimental results showed that small numbers of genes including GhBBX25, GhBBX59, GhBBX47 and GhBBX34 were expressed at low levels during these periods (Fig. 4). On the other hand, GhBBX58, GhBBX63, GhBBX49, GhBBX51, GhBBX41, GhBBX60 were expressed at lower levels at 6 DPA, 12 DPA and 18 DPA. GhBBX41 and GhBBX54 had low expression at 24 DPA, but there were high expression levels at other periods (Fig. 4). On the contrary, GhBBX25 had high expression at 18 DPA, but the expression of GhBBX25 was lower at other periods. GhBBX37 had the same expression level at these four periods. The expression of GhBBX55 was higher at 18 DPA and 24 DPA than that at other two periods (Fig. 4).
DMACA staining of transgenic Arabidopsis seeds
In Arabidopsis, AtBBX21 belongs to the structural group IV of the AtBBX gene family. The previous study desmonstrated that the mutation of the second B-box of AtBBX21 reduced the expression of CHS, CHI, F3H, F3'H, DFR and LDOX, while the mutantion of the first B-box were not significantly different from the overexpressed plants in Arabidopsis. In this study, the eukaryotic expression vectors of GhBBX27 and GhBBX33, which belong to the structural group IV similar to AtBBX21, were constructed and transformed into Arabidopsis. These seeds of transgenic plants were dyed in DMACA reagent to observe the color of the seed coat. The results displayed that the seed coat colors of transgenic Arabidopsis with GhBBX27 and GhBBX33 were dark, and the seed color of wild type Arabidopsis was a light color (Fig. 5). The color of the Arabidopsis seed coat was caused by the accumulation of proanthocyanidins in the seed coat. The transgenic Arabidopsis seed coats of GhBBX27 and GhBBX33 had a darker color than the wild type. This suggests that GhBBX27 and GhBBX33 may have an effect on the accumulation of proanthocyanidins.