Genome-Wide Identification and Classification of Arabinogalactan Proteins Gene Family in Gossypium Species and GhAGP50 Increases Numbers of Epidermal Hairs in Arabidopsis

Abstract

Arabinogalactan proteins (AGPs) constitute a diverse class of hydroxyproline-rich glycoproteins implicated in various aspects of plant growth and development. However, their functional characterization in cotton (Gossypium spp.) remains limited. As a globally significant economic crop, cotton serves as the primary source of natural fiber, making it essential to understand the genetic mechanisms underlying its growth and development. This study aims to perform a comprehensive genome-wide identification and characterization of the AGP gene family in Gossypium spp., with a particular focus on elucidating their structural features, evolutionary relationships, and functional roles. A genome-wide analysis was conducted to identify AGP genes in Gossypium spp., followed by classification into distinct subfamilies based on sequence characteristics. Protein motif composition, gene structure, and phylogenetic relationships were examined to infer potential functional diversification. Subcellular localization of a key candidate gene, GhAGP50, was determined using fluorescent protein tagging, while gene expression patterns were assessed through β-glucuronidase (GUS) reporter assays. Additionally, hormonal regulation of GhAGP50 was investigated via treatments with methyl jasmonate (MeJA), abscisic acid (ABA), indole-3-acetic acid (IAA), and gibberellin (GA). A total of 220 AGP genes were identified in Gossypium spp., comprising 19 classical AGPs, 28 lysine-rich AGPs, 55 AG peptides, and 118 fasciclin-like AGPs (FLAs). Structural and functional analyses revealed significant variation in gene organization and conserved motifs across subfamilies. Functional characterization of GhAGP50, an ortholog of AGP18 in Arabidopsis thaliana, demonstrated its role in promoting epidermal hair formation in leaves and stalks. Subcellular localization studies indicated that GhAGP50 is targeted to the nucleus and plasma membrane. GUS staining assays revealed broad expression across multiple tissues, including leaves, inflorescences, roots, and stems. Furthermore, hormonal treatment experiments showed that GhAGP50 expression is modulated by MeJA, ABA, IAA, and GA, suggesting its involvement in hormone-mediated developmental processes. This study presents a comprehensive genome-wide analysis of the AGP gene family in cotton, providing new insights into their structural diversity and functional significance. The identification and characterization of GhAGP50 highlight its potential role in epidermal hair formation and hormonal regulation, contributing to a deeper understanding of AGP functions in cotton development. These findings offer a valuable genetic resource for future research aimed at improving cotton growth and fiber quality through targeted genetic manipulation.

Keywords: arabinogalactan protein; cotton; functional characterization; gene regulation.

Figure 1

Phylogenetic and Structural Analysis of AGP Genes in G. hirsutum. (A) Phylogenetic tree illustrating the evolutionary relationships among AGP genes in G. hirsutum, con-structed using the Maximum Likelihood method with bootstrap values to indicate confidence levels. Different clades are highlighted in distinct colors to represent major evolutionary groups. (B) Conserved motif analysis displaying the distribution and arrangement of key motifs among AGP proteins. Motifs were identified using MEME, and their positions within each gene sequence are visualized, revealing similarities and differences in domain composition among AGP family members. (C) Gene structure analysis showing the exon-intron organization of AGP genes in G. hirsutum. Exons, introns, and untranslated regions (UTRs) are represented by different symbols and colors, providing insight into the structural diversity and evolutionary conservation of these genes.

Figure 2

Phylogenetic analysis of AGP gene family among the four cotton species G. hirsutum, G. arboreum, G. raimondii, and G. barbadense.

Figure 3

Duplication events analysis of AGP genes in cotton. Collinear blocks are symbolized by grey lines, while duplicated gene pairs are highlighted with blue lines.

Figure 4

Clustering of orthologous genes and duplication events analysis of AGP genes in cotton. (A) The Venn diagram of syntenic AGP genes. (B) Number of orthologous genes. (C) Number of elements shared. (D) Synonymous and nonsynonymous ratios of AGP gene family. (E) The evolutionary time of 146 duplicated gene pairs. Ka, nonsynonymous substitution rate; Ks, synonymous substitution rate; Mya, million years ago.

Figure 5

The relative expression levels of GhAGP50 with MeJA, ABA, GA, and IAA treatment. * p < 0.05; ** p < 0.01.

Figure 6

Subcellular localization of GhAGP50. (A,B) GhAGP50-GFP fusion protein was transiently observed in onion epidermal cells. Scale bar = 100 μm.

Figure 7

Tissue-specific expression of GhAGP50–GUS transgenic plants. Scale bar = 1 mm.

Figure 8

Characterization of over-expressed GhAGP50 in Arabidopsis, comparing trichomes on stalks (A) and trichomes on leaves (B) between wild-type and transgenic Arabidopsis. Scale bars = 2 cm. (C) Comparing root lengths among wild-type, mutant, and transgenic Arabidopsis. Scale bar = 1 cm. * p < 0.05; ** p < 0.01.