Comprehensive analysis of the Spartina alterniflora WD40 gene family reveals the regulatory role of SaTTG1 in plant development

Abstract

Introduction: The WD40 gene family, prevalent in eukaryotes, assumes diverse roles in cellular processes. Spartina alterniflora, a halophyte with exceptional salt tolerance, flood tolerance, reproduction, and diffusion ability, offers great potential for industrial applications and crop breeding analysis. The exploration of growth and development-related genes in this species offers immense potential for enhancing crop yield and environmental adaptability, particularly in industrialized plantations. However, the understanding of their role in regulating plant growth and development remains limited.

Methods: In this study, we conducted a comprehensive analysis of WD40 genes in S. alterniflora at the whole-genome level, delving into their characteristics such as physicochemical properties, phylogenetic relationships, gene architecture, and expression patterns. Additionally, we cloned the TTG1 gene, a gene in plant growth and development across diverse species.

Results: We identified a total of 582 WD40 proteins in the S. alterniflora genome, exhibiting an uneven distribution across chromosomes. Through phylogenetic analysis, we categorized the 582 SaWD40 proteins into 12 distinct clades. Examining the duplication patterns of SaWD40 genes, we observed a predominant role of segmental duplication in their expansion. A substantial proportion of SaWD40 gene duplication pairs underwent purifying selection through evolution. To explore the functional aspects, we selected SaTTG1, a homolog of Arabidopsis TTG1, for overexpression in Arabidopsis. Subcellular localization analysis revealed that the SaTTG1 protein localized in the nucleus and plasma membrane, exhibiting transcriptional activation in yeast cells. The overexpression of SaTTG1 in Arabidopsis resulted in early flowering and increased seed size.

Discussion: These outcomes significantly contribute to our understanding of WD40 gene functions in halophyte species. The findings not only serve as a valuable foundation for further investigations into WD40 genes in halophyte but also offer insights into the molecular mechanisms governing plant development, offering potential avenues in molecular breeding.

Keywords: Spartina alterniflora; TTG1; WD40; flowering time; seed size.

Figure 1

Identification and duplication analysis of the S. alterniflora WD40 gene family. (A) Chromosomal distribution and (B) segmental duplication events of SaWD40 genes. Connecting lines depict duplicated genes that together form pairs of duplicated genes. (C) The distribution values of ka, Ks, and Ka/Ks for segmental duplicate gene pairs. (D) The distribution values of ka, Ks, and Ka/Ks for tandem duplicate gene pairs. These values offer crucial insights into the selection pressures and evolutionary trends associated with these gene duplicates.

Figure 2

Phylogenetic and WD40 repeat analyses in S. alterniflora. (A) Phylogenetic analysis of WD40 proteins in S. alterniflora, constructed using the neighbor-joining method in MEGA 7. The tree exhibits 12 distinct phylogenetic clades with robust bootstrap support. (B) Summary of the number of WD40 repeats in S. alterniflora.

Figure 3

Expression patterns of SaWD40 genes in various tissues using RNA-seq data. The color intensity reflects expression levels, with red indicating higher expression and blue indicating lower expression.

Figure 4

Protein structure and expression patterns of SaTTG1. (A) Sequence alignment of TTG1 proteins from S. alterniflora, Arabidopsis, rice, and maize. The blue shaded area indicates WD40 repeats. (B) Phylogenetic and conserved domain analyses of TTG1 proteins from monocot and dicot plants. Sorghum bicolor SbTTG1, AFN17366.1; Zea mays ZmTTG1, NP_001310302.1; Panicum virgatum PvTTG1, XP_039854481.1; Setaria italica SiTTG1, XP_004953461.1; Brachypodium distachyon BdTTG1, XP_003570109.1; Hordeum vulgare HvTTG1, XP_044952062.1; Triticum aestivum TaTTG1, XP_044403847.1; Triticum dicoccoides TdTTG1, XP_037447801.1; Oryza sativa OsTTG1, NP_001403759.1; Arabidopsis thaliana AtTTG1, CAC10524.1. (C) qRT-PCR analysis of SaTTG1 in various tissues. R, S, L, and F represent the root, stem, leaf, and inflorescence tissues of S. alterniflora, respectively. The values presented are expressed as means ± SD, with three biological replicates (n=3). Significant differences at P < 0.05 were determined using Duncan’s multiple range test, and are indicated by different letters. (D) Subcellular localization of the SaTTG1 protein fused with eGFP in rice protoplasts. Scale bar = 20 μm. (E) Transcriptional activity of SaTTG1 in yeast cells. Yeast cells containing pGBKT7-P53 were utilized as a positive control, while yeast cells containing pGBKT7 empty vector served as a negative control.

Figure 5

Overexpression of SaTTG1 in Arabidopsis regulates plant development. (A) PCR-based DNA genotyping of the transgenic lines using specific primers. (B) Relative expression levels of the transgenic lines assessed by qRT-PCR, normalized against the expression of the internal control AtEF1α;A4. (C, D) Phenotypes of Col-0 and transgenic lines at 21 (C), Scale bar = 1 cm) and 28 days (D), Scale bar = 5 cm), respectively. (E) Statistical analysis of the number of rosette leaves on the 28th day. (F) Flowering time statistics. (G–J) Seed phenotypes of Col-0 and transgenic lines, including (G) seed phenotype photo (Scale bar = 1 mm), (H) Seed length statistics (n = 30), (I) Seed width statistics (n = 30), and (J) 1000-seed weight statistics (n = 6). Error bars represent the standard deviation, and significance tests were conducted using Student’s t-tests.