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Literature Sharing | Genome-wide analysis of the TsBLH gene family reveals TsBLH4 involved the regulation of abiotic stresses by interacting with KNOX6 in Toona sinensis
Release time:
2025-03-07
BLH1-like homeobox (BLH) transcription factors are widely distributed in plants and are a subfamily of the three-amino-acid loop extension (TALE) family. They play pivotal roles in plant growth and development processes and mediate plant responses to abiotic stress. However, recent studies on the function of BLHs have primarily focused on model plants or crops. Here, we identified 21 BLH members in the genome of Toona sinensis. The BLH gene family was divided into five subfamilies, each exhibiting variations in exon-intron distribution and motif composition. TsBLH genes exhibited tissue-specific expression, with all genes responding to salt or osmotic stresses. Notably, TsBLH4 was highly expressed in xylem and leaves and was strongly induced by both salt and osmotic stresses in leaves. Additionally, TsBLH4 is a nuclear protein that physically interacts with TsKNOX6, which is localized in the nucleus and the cytomembrane. The transient expression of TsBLH4 and TsKNOX6 genes in leaves of T. sinensis resulted in increased sensitivity to salt and enhanced tolerance to osmotic stress. These results provide a theoretical basis for the involvement of the BLH gene family in abiotic stress responses in plants.
To elucidate the evolutionary relationships of the BLH genes, we analyzed a total of 108 BLH proteins from the genomes of seven species (Fig. 1). Based on the classification of the BLH family in Arabidopsis and tomato (Hamant et al., 2010; He et al., 2022), the BLH gene family was divided into five categories. TsBLHs are distributed across all subfamilies, with 14 of them located in modules associated with Meristem Function and OFP (ovate family protein) partners, suggesting a significant role in processes such as meristem development, cell wall formation, and fruit development. In the subfamily related to Ovlue morphology, both A. thaliana and T. sinensis have only two genes, whereas O. sativa and Z. mays contain eight genes each. This disparity highlights an evolutionary divergence between dicotyledonous and monocotyledonous plants, reflecting differences in their growth and development. TsBLH4 is grouped with AtBLH2 and AtBLH4 from Arabidopsis in the subfamily associated with Leaf morphology, indicating that TsBLH4 is possibly involved in the development of leaf tissues. Interestingly, woody plants have a greater number of BLH genes compared to herbaceous plants, with the exception of tobacco. These results suggested that the BLH family might have a more prominent function in the development and adaptation of woody plants.
Fig. 1. Phylogenetic tree of BLH gene family.
The TsBLH genes are evenly distributed across 14 chromosomes and 2 chromosomal segments. Most chromosomes and chromosomal segments contain only a single TsBLH gene, except for chromosomes 5, 6, 8, 16, and 17, which each harbor two genes (Fig.2). There is evidence of clustering in the distribution of these genes. For instance, TsBLH3 and TsBLH11 are located at the same position on chromosome 8, indicating a possible gene duplication event or a closely related function (Fig.2).
Fig. 2. The distribution of the TsBLH genes located on the chromosomes in T. sinensis.
Tandem duplication and segmental duplication are two important mechanisms for the expansion of gene families. We used MCScanX and Circos to examine the homologous evolutionary relationships of BLH genes across different species (Fig. 3A). Our analysis revealed that in the BLH genes of T. sinensis, there are no tandem duplicated genes. However, we identified 19 pairs of segmental duplication events. To further investigate the presence of purifying selection, we calculated the nonsynonymous (Ka) and synonymous (Ks) substitution rates (Fig. S1). The results showed that all Ka/Ks ratios were between 0.2 and 0.3, indicating that these gene pairs have experienced purifying selection. Consequently, the expansion of the BLH gene family appears to be primarily driven by segmental duplication events and has occurred under strong purifying selection.
Fig. 3. Gene duplication and collinearity analysis of BLHs.
Through motif analysis, we identified the conserved characteristics of TsBLH proteins, recognizing a total of 12 conserved motifs (Fig. 4B). All proteins contained five conserved motifs: motif1, motif2, motif4, motif6, and motif8, indicating that these motifs are fundamental to the structure of TsBLH proteins. BLH proteins that are clustered in the same branch exhibit identical conserved motifs. To further analyze the conserved domains of BLH proteins, the BLH protein sequences were submitted to NCBI for domain analysis (Fig. 4C). The results revealed that all TsBLH proteins contain SKY, BELL, and Homeodomain conserved domains.
Fig. 4. Phylogenetic relationship, conserved motif, domain and gene structure analysis of TsBLH genes.
Located in the promoter regions of genes, cis-regulatory elements can be bound by transcription factors, leading to either the enhancement or suppression of gene expression. Therefore, predicting and analyzing the cis-regulatory elements within the promoter regions of TsBLH genes is essential for understanding their function and regulation. We retrieved a 2 kb upstream sequence of the TsBLH genes and identified a total of 30 types of elements. These elements were categorized into three primary categories: plant growth and development, plant hormone response, and stress response (Fig. 5A). Among these cis-regulatory elements, the most abundant elements are G-Box, Box4, ABRE, and MBS, which are associated with light responsiveness, organ development, abscisic acid response, and salt stress, respectively (Fig. 5B).
Fig. 5. The cis-elements of TsBLHs in T. sinensis.
The interaction network between TsBLH and TsKNOX is shown in Fig. 6, illustrating potential interactions among 10 TsBLHs and 8 TsKNOXs. Notably, TsBLH4 is observed to interact with all TsKNOXs, suggesting that TsBLH4 might function as a central transcriptional regulator. Additionally, TsBLH1, TsBLH2, and TsBLH3 also exhibit multiple interactions with other proteins. It is noteworthy that the potential interactions between TsBLHs and TsKNOXs suggest these proteins may execute their gene functions through mutual interactions.
Fig. 6. The protein–protein interaction network of TsBLHs and TsKNOXs.
To further explore the expression levels of the BLH gene family in various tissues of T. sinensis, qRT-PCR analysis was conducted. TsBLH2, TsBLH16, and TsBLH17 exhibited relatively low expression levels, resulting in undetectable Ct values. Subsequently, a heatmap was generated using the qRT-PCR data to visually represent the expression patterns (Fig. 7).
Fig. 7. The expression profiles of TsBLH genes.
TsBLH4 was predicted to be localized in the nucleus. To validate this result, we performed protein subcellular localization in the N. benthamiana leaves. We found that TsBLH4-GFP signal colocalized with mCherry signals, indicating that TsBLH4 is localized in the nucleus (Fig. 8A). We used 35S-GFP as a control, which was detected throughout the entire cell.
To determine whether the TsBLH4 gene possesses transcriptional self-activation activity, both full-length and truncated CDS sequences were cloned into pGBKT7 vectors (Fig. 8B). These constructs, along with a pGADT7 empty vector, were co-transformed into Y2H yeast strains. The transformants were then plated on selective media containing X-α-gal (SD/-Leu/-Trp) and more stringent selective media (SD/-Leu/-Trp/-His/-Ade) for verification (Fig. 8C).
Fig. 8. TsBLH4 is a nuclear protein and functions as a transcriptional activator.
To validate the function of TsBLH4, we conducted transient transformation experiments to evaluate the osmotic stress and salt tolerance capabilities in T. sinensis leaves (Fig. 9A).
Fig. 9. TsBLH4 exhibits the sensitivity to salt stress and tolerance to osmotic stress
A yeast library screening identified nine potential interacting proteins, including TsKNOX6 (Maker00020705) (Fig. S6 and Table S3). Furthermore, PPI network analysis also suggested a possible interaction between TsBLH4 and TsKNOX6 (Fig. 6). Three methods were employed to investigate the interaction between TsBLH4 and TsKNOX6. In the Y2H assay, yeast cells containing both TsBLH4 and TsKNOX6 were able to grow on the stringent selective media (SD/-Leu/-Trp/-His/-Ade), whereas those with TsBLH4-pGBKT7 and an empty vector pGADT7 could not, indicating an interaction between TsBLH4 and TsKNOX6 (Fig. 10A).
Fig. 10. TsBLH4 interacts with TsKNOX6.
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