Genome-Wide Analysis of BBX Gene Family in Three Medicago Species Provides Insights into Expression Patterns under Hormonal and Salt Stresses

Abstract

BBX protein is a class of zinc finger transcription factors that have B-box domains at the N-terminus, and some of these proteins contain a CCT domain at the C-terminus. It plays an important role in plant growth, development, and metabolism. However, the expression pattern of BBX genes in alfalfa under hormonal and salt stresses is still unclear. In this study, we identified a total of 125 BBX gene family members by the available Medicago reference genome in diploid alfalfa (Medicago sativa spp. Caerulea), a model plant (M. truncatula), and tetraploid alfalfa (M. sativa), and divided these members into five subfamilies. We found that the conserved motifs of BBXs of the same subfamily reveal similarities. We analyzed the collinearity relationship and duplication mode of these BBX genes and found that the expression pattern of BBX genes is specific in different tissues. Analysis of the available transcriptome data suggests that some members of the BBX gene family are involved in multiple abiotic stress responses, and the highly expressed genes are often clustered together. Furthermore, we identified different expression patterns of some BBX genes under salt, ethylene, salt and ethylene, salicylic acid, and salt and salicylic acid treatments, verified by qRT-PCR, and analyzed the subcellular localization of MsBBX2, MsBBX17, and MsBBX32 using transient expression in tobacco. The results showed that BBX genes were localized in the nucleus. This study systematically analyzed the BBX gene family in Medicago plants, which provides a basis for the study of BBX gene family tolerance to abiotic stresses.

Keywords: BBX; alfalfa; gene expression; stress response; transcription factors.

Figure 1

Phylogenetic relationships of BBXs retrieved in the M. sativa spp. Caerulea, M. truncatula, and M. sativa. The tree was constructed by Clustal W and MEGA X using the neighbor-joining method with 1000 bootstrap repetitions. The BBXs were classified and displayed in different groups by uniquely colored outer rings. The green triangle represents the BBXs in M. sativa spp. Caerulea; the yellow–brown circle represents the BBXs in M. truncatula; the red five-pointed star represents the BBXs in M. sativa. The number on the node in the phylogenetic tree represents the percentage of trustworthiness of the branch in the bootstrap validation.

Figure 2

Chromosomal localization analysis of BBX in M. sativa spp. Caerulea, M. truncatula, and M. sativa. (A) The orange bar indicates the M. sativa spp. Caerulea chromosomes; (B) the pink bar indicates the M. truncatula chromosomes; (C) the blue bar indicates the M. sativa chromosomes. The gene on each chromosome is highlighted in red. The scale bar on the left indicates the chromosome lengths (Mb).

Figure 3

The synteny analysis of BBXs in the genomes of M. sativa spp. Caerulea, M. truncatula, and M. sativa. (A) The collinear relationship of BBX genes in M. sativa spp. Caerulea; (B) the collinear relationship of BBX genes in M. truncatula; (C) the collinear relationship of BBX genes in M. sativa. The BBX collinear gene pairs are represented by different colored lines. The outermost circle shows the chromosome number. The information represented by each circle in the figure from inside out is gene density, GC content, chromosome length scale, and chromosome name in order.

Figure 4

The synteny analysis of BBXs in the genomes between the M. sativa spp. Caerulea, M. truncatula, and M. sativa. The red lines represent homologous gene pairs between them.

Figure 5

Expression profiles of MsBBX genes in different tissues of M. sativa. Heat maps reflect the transcripts per million (TPM) of MsBBXs. The color bar from blue to red indicates relative expression levels from lower to higher, respectively.

Figure 6

Expression profiles of MsBBX genes under different abiotic stresses and hormone treatments of M. sativa. Heat maps reflect the transcripts per million (TPM) of MsBBXs. The color bar from blue to red indicates relative expression levels from lower to higher, respectively.

Figure 7

The expression profiles of 15 selected MsBBX genes in response to NaCl, ETH, and NaCl and ETH treatment in M. sativa by using qRT-PCR. Data are the average of three independent biological samples ± SD and vertical bars indicate standard deviation. Different letters indicate significant differences at p < 0.05, as determined by Duncan’s multiple range tests.

Figure 8

The expression profiles of 15 selected MsBBX genes in response to NaCl, SA, and NaCl and SA treatment in M. sativa by using qRT-PCR. Data are the average of three independent biological samples ± SD and vertical bars indicate standard deviation. Different letters indicate significant differences at p < 0.05, as determined by Duncan’s multiple range tests.

Figure 9

Subcellular localization of MsBBX2, MsBBX17, and MsBBX32. MsBBX:GFP fusion proteins were transiently expressed in Nicotiana benthamiana leaves, and their localization was determined using confocal microscopy. The nucleus was visualized with mCherry-labeled nuclear markers. Bar = 50 µm.