Identification and Characterization of the BBX Gene Family in Bambusa pervariabilis × Dendrocalamopsis grandis and Their Potential Role under Adverse Environmental Stresses

Abstract

Zinc finger protein (ZFP) transcription factors play a pivotal role in regulating plant growth, development, and response to biotic and abiotic stresses. Although extensively characterized in model organisms, these genes have yet to be reported in bamboo plants, and their expression information is lacking. Therefore, we identified 21 B-box (BBX) genes from a transcriptome analysis of Bambusa pervariabilis × Dendrocalamopsis grandis. Consequently, multiple sequence alignments and an analysis of conserved motifs showed that they all had highly similar structures. The BBX genes were divided into four subgroups according to their phylogenetic relationships and conserved domains. A GO analysis predicted multiple functions of the BBX genes in photomorphogenesis, metabolic processes, and biological regulation. We assessed the expression profiles of 21 BBX genes via qRT-PCR under different adversity conditions. Among them, eight genes were significantly up-regulated under water deficit stress (BBX4, BBX10, BBX11, BBX14, BBX15, BBX16, BBX17, and BBX21), nine under salt stress (BBX2, BBX3, BBX7, BBX9, BBX10, BBX12, BBX15, BBX16, and BBX21), twelve under cold stress (BBX1, BBX2, BBX4, BBX7, BBX10, BBX12, BBX14, BBX15, BBX17, BBX18, BBX19, and BBX21), and twelve under pathogen infestation stress (BBX1, BBX2, BBX4, BBX7, BBX10, BBX12, BBX14, BBX15, BBX17, BBX18, BBX19, and BBX21). Three genes (BBX10, BBX15, and BBX21) were significantly up-regulated under both biotic and abiotic stresses. These results suggest that the BBX gene family is integral to plant growth, development, and response to multivariate stresses. In conclusion, we have comprehensively analyzed the BDBBX genes under various adversity stress conditions, thus providing valuable information for further functional studies of this gene family.

Keywords: B-box gene; Bambusa pervariabilis × Dendrocalamopsis grandis; abiotic stress; bioinformatics analyses; biotic stress.

Figure 1

The phylogenetic tree, protein domain, conserved motifs and their sequence information within the BDBBX protein family, and the phylogenetic tree of relationships between members of the BBX protein family in Arabidopsis thaliana, Oryza sativa, and Bambusa pervariabilis × Dendrocalamopsis grandis. (A) Phylogenetic tree, protein structural domains, conserved motifs of BDBBX protein; and (B) Sequence information of conserved motifs of BDBBX protein. Sequence information includes each conserved sequence’s length, the conservation degree, and the amino acid’s sharing or substitution. Each motif has a corresponding axis, with the horizontal axis representing the corresponding position of each amino acid on the pattern and the vertical axis representing the sections. (C) Phylogenetic tree of the neighbor-joining method for the BBX proteins of Bambusa pervariabilis × Dendrocalamopsis grandis, Arabidopsis thaliana, and Oryza sativa. Classes of different colors represented different subfamilies of Bambusa pervariabilis × Dendrocalamopsis grandis.

Figure 2

Secondary and tertiary structures of BBX proteins. (A) is Group I, (B) is Group III, (C) is Group IV, and (D) is Group V. The sequence of the protein is segmented from n to c, and the color transitions from cool (blue) to warm (red), with different colors representing different helices and folding regions.

Figure 3

GO annotation of the BDBBX gene. (A) shows the Venn diagram of the distribution of BDBBX genes in the biological process (BP), molecular function (MF), and cellular component (CC) functional categories. (B) shows the subcategories of the GO classification of the BDBBX gene (Level 2), where the x-axis represents the number of genes, and the y-axis represents the name of the GO subcategory function.

Figure 4

BDBBX gene family protein–protein interaction network. The connecting lines of protein interactions have different meanings due to their different colors. Yellow represents text mining, black represents co-expression, purple represents protein physiology, and pink represents experimentally determined.

Figure 5

Changes in the expression of 21 BDBBX genes over time in three varieties of hybrid bamboo under water deficit stress. Histogram showing the changes in the expression of 21 BDBBX genes over time in three varieties of hybrid bamboo under water deficit stress. The hybrid bamboo was stressed using a 20% concentration of PEG-6000 solution. Leaves of the plants were taken after 0 h, 6 h, 12 h, and 24 h, and the expression of the hybrid bamboo BBX genes was detected by qPCR testing. The relative expression of the target genes was calculated using the 2^−∆∆Ct method. The horizontal coordinates indicate each sampling time point of the experiment, and the vertical coordinates indicate the relative expression levels. Different lowercase letters indicate the significance of differences between treatments at the 0.005 level (p < 0.05).

Figure 6

Changes in the expression of 21 BDBBX genes over time in three varieties of hybrid bamboo under salt stress. Histogram showing the changes in the expression of 21 BDBBX genes over time in three varieties of hybrid bamboo under salt stress. The hybrid bamboo was stressed using 200 mM NaCl solution. Leaves of the plants were taken after 0 h, 6 h, 12 h, and 24 h, and the expression of the hybrid bamboo BBX genes was detected by qPCR testing. The relative expression of the target genes was calculated using the 2^−∆∆Ct method. The horizontal coordinates indicate each sampling time point of the experiment, and the vertical coordinates indicate the relative expression levels. Different lowercase letters indicate the significance of differences between treatments at the 0.005 level (p < 0.05).

Figure 7

Changes in the expression of 21 BDBBX genes over time in three varieties of hybrid bamboo under cold stress. Histogram showing the changes in the expression of 21 BDBBX genes over time in three varieties of hybrid bamboo under cold stress. Transfer the hybrid bamboo to a cooler (SANYO) at 10 °C for stress. Leaves of the plants were taken after 0 h, 6 h, 12 h, and 24 h, and the expression of the hybrid bamboo BBX genes was detected by qPCR testing. The relative expression of the target genes was calculated using the 2^−∆∆Ct method. The horizontal coordinates indicate each sampling time point of the experiment, and the vertical coordinates indicate the relative expression levels. Different lowercase letters indicate the significance of differences between treatments at the 0.005 level (p < 0.05).

Figure 8

Changes in the expression levels of the 21 BDBBX genes of the three hybrid bamboo varieties under disease infestation conditions with a time of infestation. The histograms show the changes in the expression of the 21 BDBBX genes over time in the different hybrid bamboo varieties under disease infestation. Hybrid bamboos were infested by injection of the spore suspension of the pathogen, and leaves of the plants were taken after 0 d, 3 d, 5 d, 7 d, and 10 d. The expression of the hybrid bamboo BBX genes was detected by qPCR testing. The relative expression of the target genes was calculated using the 2^−∆∆Ct method. The horizontal coordinates indicate each sampling time point of the experiment, and the vertical coordinates indicate the relative expression levels. Different lowercase letters indicate the significance of differences between treatments at the 0.005 level (p < 0.05).

Figure 9

The multiple sequence alignments of BBX proteins from three different species.