Genomic Organization, Phylogenetic and Expression Analysis of the B-BOX Gene Family in Tomato

Abstract

The B-BOX (BBX) proteins encode a class of zinc-finger transcription factors possessing one or two B-BOX domains and in some cases an additional CCT (CO, CO-like and TOC1) motif, which play important roles in regulating plant growth, development and stress response. Nevertheless, no systematic study of BBX genes has undertaken in tomato (Solanum lycopersicum). Here we present the results of a genome-wide analysis of the 29 BBX genes in this important vegetable species. Their structures, conserved domains, phylogenetic relationships, subcellular localizations, and promoter cis-regulatory elements were analyzed; their tissue expression profiles and expression patterns under various hormones and stress treatments were also investigated in detail. Tomato BBX genes can be divided into five subfamilies, and twelve of them were found to be segmentally duplicated. Real-time quantitative PCR analysis showed that most BBX genes exhibited different temporal and spatial expression patterns. The expression of most BBX genes can be induced by drought, polyethylene glycol-6000 or heat stress. Some BBX genes were induced strongly by phytohormones such as abscisic acid, gibberellic acid, or ethephon. The majority of tomato BBX proteins was predicted to be located in nuclei, and the transient expression assay using Arabidopsis mesophyll protoplasts demonstrated that all the seven BBX members tested (SlBBX5, 7, 15, 17, 20, 22, and 24) were localized in nucleus. Our analysis of tomato BBX genes on the genome scale would provide valuable information for future functional characterization of specific genes in this family.

Keywords: BBX; gene expression; phylogenetic analysis; subcellular localization; tomato.

Figure 1

Structure of the tomato BBX proteins. Numbers indicate amino acid position of the corresponding conserved domains. The red, blue rectangles and green pentagons indicate the B-BOX1, B-BOX2 and CCT domain, respectively. The scale bar represents 100 amino acids.

Figure 2

Phylogenetic analysis of the tomato BBX family. The trees shown are based on the alignments of the protein sequences of the full length (A), the B-BOX 1 domain (B), and two B-BOX plus the CCT domains (C). The bootstrap values are indicated at each node. The scale bar represents 0.1 amino acid substitutions per site. The members marked in black triangle contain two B-BOX and one CCT domains.

Figure 3

Gene structure of the tomato BBX family generated from GSDS. The yellow block means the coding sequence (CDS), the blue block means the upstream or downstream of the genes, and the black line indicates the intron. The scale bar indicates the length of the DNA sequences.

Figure 4

Chromosome distribution and duplication events of tomato BBX genes. Chromosomal mapping was based on the physical position (Mb) in 12 tomato chromosomes. The chromosome number is indicated at the top of each bar. The positions of the tomato BBX genes in the chromosomes were obtained from Sol Genomics Network database (SGN, https://www.sgn.cornell.edu). 7 pairs of paralogous gene connected with black dotted lines represent segmental duplication. The black blocks indicate the positions of the centromeres in chromosomes, respectively. Scale represents 10 Mb chromosomal distance.

Figure 5

Expression profiles of tomato BBX genes in various organs. The relative expression data were log2 transformed with R software, and a cluster dendrogram is shown on the left of the heat map. Blocks with blue colors indicate decreased and red ones indicate increased transcription levels. Rt, root; YS, young stem; YL, young leaf; Fl, flower; IM, immature fruit; MG, mature green fruit; BR, breaker fruit; YR, yellow ripe fruit; RR, red ripe fruit.

Figure 6

Expression profiles of tomato BBX genes under treatments of exogenous hormones in hydroponic culture. The relative expression data were log2 transformed with R software, and a cluster dendrogram is shown on the left of each heat map. Blocks with colors indicate decreased (blue) or increased (red) transcript level relative to the corresponding control (Plants grown in hydroponic solution at the same time without hormone treatments). (A–H) represent the gene expression patterns of BBX genes detected using real-time PCR under eight different hormone treatments. CK, GA, BR, ETH, ABA, 6-BA, SA, MeJ, and IAA represent seedlings treated with water, 5 μM gibberellic acid, 5 μM brassinosteroids, 50 μM ethephon, 5 μM abscisic acid, 5 μM 6-benzylaminopurine, 5 μM salicylic acid, 5 μM methyl jasmonate, and 5 μM auxin, respectively. The numbers 0.5, 1, 2, 6, 12, and 24 indicate the time (hour) after treatments, and 15 min represents 15 min after treatment.

Figure 7

Expression patterns of tomato BBX genes under different abiotic stresses. The relative expression data were log2 transformed with R software, and a cluster dendrogram is shown on the left of each heat map. Blocks with blue colors indicate decreased and red ones indicate increased transcription levels. (A) shows the relative transcription level of BBX genes under different stress treatments in hydroponic culture condition. PEG, NaCl and MV represent seedlings treated with 10% polyethylene glycol-6000, 100 mM NaCl, and 5 μM Methylviologen, respectively. The numbers 0.5, 1, 2, 6, 12, and 24 indicate the time (hour) after treatments, and 15 min represents 15 min after treatment. Plants without stress (CK) at the same time are served as the control. (B) represents the gene expression patterns of the BBX genes treated with heat, cold, and dehydration stresses. For the heat stress, samples were collected at 3, 6, 12, and 24 h after 42°C treatment, and for the cold stress, samples were obtained at 1.5, 6, 12, and 24 h after 4°C treatment. Samples were taken at 0.5, 1, 1.5, 2, and 2.5 h after drought treatment (imitated with air drying the plants).

Figure 8

Subcellular localization of seven GFP-fused tomato BBX proteins. The plant expression vectors of green fluorescent protein (GFP) fused BBX proteins or control vector (pGWB451) were transformed into Arabidopsis protoplasts. The fluorescence was observed by confocal microscopy 24 h later. Nuclei were visualized by co-transformation of a cyan fluorescent protein (CFP) fused nucleus marker, Ghd7. Scale bar, 5 μm.