Genome-Wide Identification of the Whirly Gene Family and Its Potential Function in Low Phosphate Stress in Soybean (Glycine max)

Abstract

The Whirly (WHY) gene family, functioning as transcription factors, plays an essential role in the regulation of plant metabolic responses, which has been demonstrated across multiple species. However, the WHY gene family and its functions in soybean remains unclear. In this paper, we conducted genome-wide screening and identification to characterize the WHY gene family. Seven WHY members were identified and randomly distributed across six chromosomes. The phylogenetic evolutionary tree of WHY genes in soybean and other species was divided into five clades. An in-depth analysis revealed that segmental duplications significantly contributed to the expansion of GmWHYs, and the GmWHY gene members may have experienced evolutionary pressure for purifying selection in soybeans. The analysis of promoter Cis-elements in GmWHYs suggested their potential significance in addressing diverse stress conditions. The expression patterns of GmWHYs exhibited tissue-specific variations throughout the different stages of soybean development. Additionally, six GmWHY genes exhibited different responses to low phosphate stress. These findings will provide a theoretical basis and valuable reference for the future exploration of WHY gene function.

Keywords: expansion; expression patterns; haplotype; phosphate deficiency; whirly.

Figure 1

Comparison of the WHY gene family. (A) Phylogenetic analysis of the WHY gene family. The phylogenetic tree was constructed for the WHY gene family using 37 WHY genes from various plant species, including A. thaliana (At), O. sativa (Os), M. truncatula (Mt), M. sativa (Ms), L. japonicus (Lj), P. coccineus (Pc), G. max (Gm), and G. soja (Gs). The classification of the WHY gene family into subfamilies was denoted as I, II, III, IV, and V. (B) Positions and synteny of GmWHY genes. The duplicated GmWHY gene pairs were connected by red lines. (C) Synteny patterns of the WHY genes between G. max and A. thaliana. (D) Synteny patterns of the WHY genes between G. max and G. soja. The red lines were used to emphasize the syntenic WHY gene pairs between soybean and other species.

Figure 2

Structure of GmWHYs. (A) The phylogenetic tree, gene structure, domains, and motifs of GmWHYs. Left panel: exons and introns are represented by red color boxes and gray lines, respectively, and the domains are represented by differently colored boxes. Right panel: ten motifs are represented in differently colored boxes and were predicted by MEME [26]. The sizes of exons and introns are proportional to their sequence lengths. (B) Predicted protein structure of GmWHYs. N: and C represent the N-terminus and C-terminus of the protein, respectively.

Figure 3

Cis-elements in the GmWHY gene promoter regions. The arrangement of the cis-regulatory elements within the 2000 bp upstream genetic regions of the seven identified GmWHYs is represented by colored boxes, each indicating a different cis-element.

Figure 4

Haplotypes of GmWHY genes among the natural soybean population. Green, yellow, purple, and blue bars represent 5′UTR, introns, exons, and 3′UTR.

Figure 5

Analysis of GmWHY expression in various soybean tissues. Evaluation of GmWHY expression in different tissues during soybean development using publicly available RNA-seq data. The heatmap depicts log2-normalized RPKM values to represent gene expression levels. DAFs denote days after flowering.

Figure 6

Expression analyses of GmWHYs in the leaves and roots of the cultivar Williams 82 under different Pi treatment levels. Data were normalized to the GmTubulin gene, and columns and error bars represent the means ± standard deviation (SD) of three independent biological replicates. Differences were evaluated using the two-tailed Student’s t-test (*** p < 0.001, ** p < 0.01, * p < 0.05, ns = no differences). LP, low Pi supply (5 μM, Pi); NP, normal Pi supply (500 μM, Pi).