The genomic data, encompassing DNA, complete coding sequence (CDS), protein sequence, and positional annotations of S. alterniflora, were procured from our compiled genome. To identify WD40 proteins in S. alterniflora, we initially searched for sequences exhibiting the characteristic WD40 motif (PF00400) within the S. alterniflora protein sequences using HMMER 3.0. The putative SaWD40 proteins were validated using SMART (Letunic et al., 2021) and NCBI’s conserved domain database (Marchler-Bauer et al., 2015). Finally, only the proteins containing the WD40 repeat were retained for subsequent analysis.

To examine their physical attributes, all SaWD40 sequences were assessed using EXPASY to record the number of amino acids and molecular.

To construct a phylogenetic tree for the SaWD40s, we first performed multiple sequence alignment of the full-length SaWD40 protein sequences using the T-COFFEE program (Magis et al., 2014). Subsequently, we employed the Neighbor-Joining (NJ) method in MEGA 7 to build the phylogenetic tree, with a Bootstrap value of 1000 and other default parameters (Kumar et al., 2016). Finally, we utilized Evolview v3 (Subramanian et al., 2019) to visualize the generated phylogenetic tree.

To analyze gene duplication events in SaWD40 genes, we employed MCScanX (Wang et al., 2012) with default settings to identify various gene duplicates, including whole-genome and tandem duplications. The chromosomal locations and duplicated WD40 gene pairs were visualized using TBtools software (Chen et al., 2020). Evolutionary dynamics of these gene pairs were assessed by calculating Ka/Ks values using KaKs_Calculator v 2.0 (Zhang, 2022). The divergence time was determined using the formula T = Ks/2λ, where Ks signifies the synonymous substitutions per site, and λ represents the rate of divergence for nuclear genes in plants. For monocot plants, the adopted λ value fell within the range of 5.1–7.1×10^–9 synonymous substitutions per site per year.

To conduct tissue-specific expression analysis, S. alterniflora seedlings were grown in an artificial chamber with a temperature of 24/22°C (day/night) and a photoperiod of 16 hours/8 hours (day/night). During the flowering stage, roots, stems, leaves, and inflorescences were collected for analysis. Immediately after sampling, the samples were rapidly frozen in liquid nitrogen and stored at -80°C for RNA extraction.

Total RNA was extracted from plants using the RNAsimple Total RNA Kit (Tiangen, Beijing) following the manufacturer’s instructions. The synthesis of the first-strand cDNA was performed using the FastKing RT Kit (Tiangen, Beijing), and the experimental steps from the kit’s manual were followed. qRT-PCR was conducted using the SYBR Green Pro Taq HS Kit (Tiangen, Beijing), following the manufacturer’s protocol. Data was normalized by the expression of GAPDH in S. alterniflora and AtEF1α4 in Arabidopsis. Each group of experiments was repeated in three biological replicates, and the relative expression levels were determined using the 2^–ΔΔCt analysis method (Livak and Schmittgen, 2001). DNA extraction was carried out using the CTAB method. The primer sequences used in this study are listed in Supplementary Table 1 .

The SaTTG1 CDS was efficiently amplified by RT-PCR and cloned into the pCAMBIA-1300 vector. Transgenic Arabidopsis Columbia-0 lines were obtained using the floral dip method (Clough and Bent, 1998). Positive lines were screened on 1/2 MS solid medium with 40mg/L Hygromycin B and genotype verified by PCR. Mature seeds from wild-type and transgenic lines were imaged using an SMZ25 microscope (Nikon).

To ascertain the subcellular localization of SaTTG1, the CDS of SaTTG1 was cloned into PAN580-GFP and introduced into rice (Oryza sativa) protoplasts. Subsequently, subcellular localization was observed using a confocal laser scanning microscope (Zeiss LSM 980 with Airysca, Germany). To assess transcriptional activity, the SaTTG1 CDS was ligated into the pGBKT7 vector for fusion expression. The resulting pGBKT7-SaTTG1 was introduced into Y2H, which were then serially diluted 10-fold and spotted on SD/-Trp plates. Transcriptional activity was evaluated by monitoring yeast growth on SD/-Trp-Ade-His plates. The pGBKT7 vector served as a negative control, with pGBKT7-P53 as a positive control.

Root, seed length, and width of Arabidopsis were quantified with ImageJ (Rueden et al., 2017) and analyzed in Microsoft Excel 2013. Error bars represent standard deviation, and significant differences are marked with numbers or letters.

To unravel the functional aspects of the WD40 gene family in S. alterniflora, we employed the conserved domain of WD40 proteins (PF00400) as a query in the Pfam database, conducting a comprehensive search against the S. alterniflora genome protein sequences. This exploration led to the identification of a total of 582 SaWD40 members distributed across 31 chromosomes. Chromosome 7 exhibited the highest density of SaWD40 genes, while chromosomes 19 and 31 displayed the lowest density ( Figure 1A ). The nomenclature of SaWD40 genes was assigned as SaWD40–1 to SaWD40–582 based on their respective chromosome locations ( Supplementary Table 2 ). The deduced SaWD40 DNA lengths ranged from 456 (SaWD40–276) to 52,866 bp (SaWD40–311), and protein lengths spanned from 70 (SaWD40–276) to 3,581 (SaWD40–30) amino acids (aa). The molecular weight varied between 7.44 (SaWD40–276) and 398.31 (SaWD40–30) kDa. Detailed physicochemical properties of S. alterniflora are comprehensively listed in Supplementary Table 2 .

Within the cohort of 582 SaWD40 genes, 490 genes participated in the formation of 605 segmentally duplicated gene pairs, while 20 genes contributed to 10 tandemly duplicated gene pairs ( Figure 1B ; Supplementary Table 3 ). This implies a significant role of segmental duplication in expanding the SaWD40 gene family. Further scrutiny of the synonymous (Ks) and non-synonymous (Ka) mutations in these gene pairs ( Figures 1C, D ; Supplementary Table 3 ) provided insights into the evolutionary dynamics. Moreover, the substitution rate (Ka/Ks) effectively determines positive selection pressure post-duplication, revealing evolutionary direction and selective strength in coding sequences. A Ka/Ks ratio of 1 indicates neutral selection, <1 purifying selection, and >1 positive selection. We calculated Ka, Ks, and Ka/Ks for each gene pair. The average Ka/Ks for tandem duplication SaWD40 genes (0.3875) was significantly higher than for segmental duplication genes (0.2735). Additionally, tandem and segmental duplication events were estimated to have occurred ~256.85–357.58 and ~205.48–286.06 Mya, respectively. This observation suggests the prevalence of purifying selection, indicating that these genes have encountered functional constraints and selective pressure in maintaining their essential functions.

To depict the evolutionary relationships among these 582 SaWD40 proteins, we constructed an unrooted phylogenetic tree utilizing the NJ method ( Figure 2A ; Supplementary Figure 1 ). The bootstrap values within the tree facilitated the division of these WD40 proteins into 12 clades. Clade IV boasted the largest contingent of WD40 members, encompassing 145 members, followed by 118 in Clade VII and 87 in Clade II, whereas Clade XI exhibited a more modest presence with only five members.

The SaWD40 genes exhibit a wide spectrum of WD40 repeats, ranging from 1 to 15 WD40 repeats ( Figure 2B ). Previous studies have indicated that proteins containing WD repeats typically possess 4–10 WD repeats (Van Nocker and Ludwig, 2003). In our SaWD40 protein repertoire, 459 members were identified to contain 4–10 WD repeats, while 39 members displayed more than 10 WD repeats.

We delved into the exon/intron structures of the SaWD40 genes. The analysis unveiled a variation in the number of exons within the SaWD40 gene structure, spanning from 1 to 41 ( Supplementary Table 2 ), with an average of 10.83 exons. Remarkably, SaWD40–311 boasted the largest number of exons (41), followed by SaWD40–340 with 39 exons. In contrast, 21 SaWD40 (3.6%) genes were observed to possess only one exon. The prevalence of genes with seven exons stood out, constituting the highest percentage at 8.8% (51/582) among the total gene count.

To elucidate the functional roles of WD40 genes, we conducted an analysis of their expression patterns across various tissues in the S. alterniflora transcriptome. As depicted in Figure 3 and detailed in Supplementary Table 4 , with the exception of 11 genes (FPKM < 0.1) that exhibited undetectable expression levels across different tissues – namely SaWD40–29, -74, -103, -152, -194, -220, -271, -345, -374, -392, -580 – the remaining 565 genes can be detected exhibiting expression (FPKM ≥ 0.1) in certain tissues. Three hundred sixty genes displayed ubiquitous expression across all tissues. There were nine genes that exhibit distinct tissue-specific expression patterns. For instance, SaWD40–194 exhibited exclusive expression in roots, while SaWD40–346, SaWD40–379, SaWD40–391, and SaWD40–502 were specifically expressed in developing inflorescences; SaWD40–85, SaWD40–193, SaWD40–376, and SaWD40–500 displayed specific expression in mature seeds. Furthermore, we observed that certain genes are expressed at higher levels in specific tissues compared to other tissues. For instance, eight genes (SaWD40–73, SaWD40–252, SaWD40–256, SaWD40–293, SaWD40–258, SaWD40–283, SaWD40–444, SaWD40–477) exhibited higher expression levels in maturing seeds, while eight genes (SaWD40–19, SaWD40–102, SaWD40–107, SaWD40–125, SaWD40–219, SaWD40–272, SaWD40–488, SaWD40–514) demonstrated higher expression in developing inflorescences. These findings underscore the relatively widespread expression of WD40 genes across diverse tissues.

Analysis of expression patterns revealed that WD40 genes can be detected in various tissues of S. alterniflora, indicating their involvement in diverse growth and development stages of the plant. To validate the role of WD40 genes in plant growth and development, we aimed to functionally characterize them through heterologous expression in A. thaliana. TTG1, a gene encoding a WD40 protein, has been shown to play a pivotal role in multiple aspects of plant growth and development. Its involvement spans processes such as the accumulation of seed storage reserves (Chen et al., 2015), biosynthesis of anthocyanin and proanthocyanidin (Shan et al., 2019), regulation of circadian activity, epidermal cell fate, and pigmentation (Airoldi et al., 2019). However, to date, there have been no reports on the functional roles of WD40 genes in S. alterniflora. Expression pattern analysis revealed that SaWD40–256, a homolog of A. thaliana’s TTG1 gene, is expressed at its highest level in maturing seeds. Therefore, we aimed to functionally characterize the S. alterniflora TTG1 gene through heterologous expression in A. thaliana, with the goal of gaining deeper insights into the conservation of its functional properties.

Seeking a comprehensive understanding of the function of SaTTG1 (SaWD40–256) in S. alterniflora, we isolated the gene from young seedlings of this species. As depicted in Figure 4A , SaTTG1 exhibits a high sequence similarity with TTG1 proteins from Arabidopsis, Zea mays, and Oryza sativa. These proteins share a highly conserved WD40 repeat regulatory domain. To unravel the potential functions of the SaTTG1 protein, a phylogenetic analysis was conducted using SaTTG1 and TTG1 proteins from other plants. The results suggest that the functions of SaTTG1 more closely resemble those of monocot proteins than dicot proteins ( Figure 4B ; Supplementary Figure 2 ).

Figure 4C illustrates the presence of SaTTG1 transcript in various tissues, with the lowest expression level detected in leaves. This indicates that the gene plays a widespread role in the growth and developmental processes of plants. Subcellular localization studies revealed that SaTTG1, when transiently expressed as 2×35S::SaTTG1-eGFP in rice protoplasts, exhibited a fluorescent signal exclusively in the nucleus and plasma membrane ( Figure 4D ). Furthermore, yeast cells transformed with the pGBDT7-SaTTG1 fusion construct demonstrated activation of reporter genes and survival in the selective medium SD/-Trp-Ade-His ( Figure 4E ), affirming the transcriptional activation activity of SaTTG1 in yeast.

To examine the function of SaTTG1, we introduced the overexpression construct of 35S::SaTTG1 into the Arabidopsis wild-type Col-0. Through hygromycin selection, a total of 20 independent T1 transgenic plants were acquired. Subsequently, two independent T3 homozygous Col-0 35S::SaTTG1 transgenic lines (#1 and #2) were selected and validated by PCR ( Figure 5A ). qRT-PCR analysis corroborated the substantial expression of SaTTG1 in the respective transgenic lines, with no detection in the wild-type Col-0 ( Figure 5B ). These results demonstrate that the overexpression of SaTTG1 induces early flowering in Arabidopsis, and the number of rosette leaves in overexpressing plants is reduced ( Figures 5C–F ). The seed size of the transgenic lines surpassed that of Col-0, displaying augmented seed length and width ( Figures 5G–I ). Correspondingly, the 1000-seed weight of the transgenic lines exhibited a notable increase by 9.29% and 8.10% compared to Col-0 ( Figure 5J ). These findings underscore the significant role of SaTTG1 in the regulation of plant development.