Molecular evolutionary analysis of the SHI/STY gene family in land plants: A focus on the Brassica species

Abstract

The plant-specific SHORT INTERNODES/STYLISH (SHI/STY) proteins belong to a family of transcription factors that are involved in the formation and development of early lateral roots. However, the molecular evolution of this family is rarely reported. Here, a total of 195 SHI/STY genes were identified in 21 terrestrial plants, and the Brassica species is the focus of our research. Their physicochemical properties, chromosome location and duplication, motif distribution, exon-intron structures, genetic evolution, and expression patterns were systematically analyzed. These genes are divided into four clades (Clade 1/2/3/4) based on phylogenetic analysis. Motif distribution and gene structure are similar in each clade. SHI/STY proteins are localized in the nucleus by the prediction of subcellular localization. Collinearity analysis indicates that the SHI/STYs are relatively conserved in evolution. Whole-genome duplication is the main factor for their expansion. SHI/STYs have undergone intense purifying selection, but several positive selection sites are also identified. Most promoters of SHI/STY genes contain different types of cis-elements, such as light, stress, and hormone-responsive elements, suggesting that they may be involved in many biological processes. Protein-protein interaction predicted some important SHI/STY interacting proteins, such as LPAT4, MBOATs, PPR, and UBQ3. In addition, the RNA-seq and qRT-PCR analysis were studied in detail in rape. As a result, SHI/STYs are highly expressed in root and bud, and can be affected by Sclerotinia sclerotiorum, drought, cold, and heat stresses. Moreover, quantitative real-time PCR (qRT-PCR) analyses indicates that expression levels of BnSHI/STYs are significantly altered in different treatments (cold, salt, drought, IAA, auxin; ABA, abscisic acid; 6-BA, cytokinin). It provides a new understanding of the evolution and expansion of the SHI/STY family in land plants and lays a foundation for further research on their functions.

Keywords: Cis-acting elements; SHI/STY gene family; gene duplication; land plants; molecular evolution.

Figure 1

SHI/STY genes are identified in land plants. The number of SHI/STY genes identified in each sequenced plant genome was listed in sequence. Percentages for each type are listed in parentheses. The red star marks indicate when SHI/STY appeared during the evolution of land plants. “-” indicates that there is no SHI/STY in this species.

Figure 2

Sequence logos of conserved motifs in SHI/STY proteins. This logo represents the sequence of 16 motifs. The letters represent any amino acid, and the size represents how well it has been preserved at that location. The predicted motif sequence was obtained from the MEME website based on the sequences of SHI/STYs (http://meme-suite.org/tools/meme).

Figure 3

Different motif distribution of the SHI/STY proteins and exon–intron organization in different clades. The different colorful patterning represents the relative positions of motifs on the protein sequence in the different clades. The inverted triangles with yellow, green and blue marks indicate the insertion positions of phase 0, 1 and 2 introns, respectively.

Figure 4

Chromosome distribution of BnSHI/STYs. 25 BnSHI/STYs are distributed on 19 chromosomes of Brassica napus. In addition, 3 BnSHI/STYs are not mapped to chromosomes. The duplicated BnSHI/STY gene pairs were indicated in red lines. The number of chromosomes is displayed on each chromosome.

Figure 5

The collinear relationship of SHI/STYs on the chromosome. A chromosome is represented by a bar. The green, yellow, and purple bars represent the chromosomes of Arabidopsis, B. rapa, and B. oleracea, respectively. The collinear blocks (between the genomes of B. napus and Arabidopsis, B. napus and B. rapa, B. napus and B. oleracea) were indicated in the gray lines.

Figure 6

Cis-element analysis in the promoter regions of the SHI/STY genes. The PlantCARE software was used to determine the presence of different cis-acting elements. The different cis-elements were represented in different colored boxes.

Figure 7

The tertiary spatial structure of BnSHI/STY protein in Clade 3. The eleven predicted positive selection sites were marked with arrows.

Figure 8

The protein–protein interaction network of SHI/STYs. Eleven SHI/STYs were involved in the construction of the protein–protein interaction network. Fifty-three protein–protein interactions were found between SHI/STYs and some other proteins. The colorful balls represent different proteins and the line between proteins indicates the interaction between the two proteins.

Figure 9

The expression patterns of BnSHI/STYs in different tissues. BnSHI/STYs are clustered by hierarchical clustering. The different color box represents expression quantity in different tissues, red to blue means expression lever from high to lower.

Figure 10

The expression patterns of BnSHI/STYs. (A) The expression pattern of BnSHI/STYs after infection by Sclerotinia sclerotiorum. (B) The expression pattern of BnSHI/STY in B. napus under abiotic stress. (C) The expression pattern of BnSHI/STYs in cold stress. RNA sequence data that were treated with cold shock at temperatures of chill (4°C) and freezing (4°C) were used to complete this figure, and “CK” was treated at 25°C. Genes with high expression levels are shown in red, and genes with low expression levels are shown in blue. The gray box indicates that the expression data of this gene were not found in the database.

Figure 11

Expression levels of eight BnSHI/STYs under different abiotic stresses and hormone treatments. The selected BnSHI/STYs were normalized concerning the reference gene (Actin). The X-axis corresponds to different abiotic stress treatments and hormone treatments. The values on the Y-axis represent the mean ± SD of three biological replicates. Asterias on vertical bar shows significant difference at ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001.

Figure 12

Evolution scenario of the SHI/STY gene family in land plants. In Physcomitrella patens and Selaginella oellendorffii, the SHI/STY gene family emerged. The SHI/STY gene family is formed by adding different motifs at both ends of conserved domains to form Clade 1 and Clade 3 in monocotyledons. In dicotyledons, the Clade 3 gene was further duplicated and gradually evolved into three branches through loss or acquisition of new motifs (some retained the characteristics of Clade 3, some acquired motif 8 and evolved into Clade 2, while the conserved motif in Clade 4 was lost).