Understanding the transition from embryogenesis to seed filling in Phaseolus vulgaris L. non-endospermic seeds

Abstract

Introduction: Common bean (Phaseolus vulgaris L.) is one of the most consumed grain legumes. These legumes are a major source of proteins and other important nutrients, especially in developing countries. Studying seed development in common bean is crucial for improving yield, nutrition, stress tolerance and disease resistance while promoting sustainable agriculture and food security, with its sequenced genome and available molecular tools making it an excellent research model. Despite advances in studying P. vulgaris seed development, the precise timing and molecular regulation of the transition from embryogenesis to seed filling remain poorly understood. Although P. vulgaris seeds at 10 days after anthesis (DAA) were previously characterized as being in the late embryogenesis stage, our previous studies suggested that this transition might occur earlier than 10 DAA, prompting us to investigate earlier developmental stages.

Methods: To accomplish this goal, we conducted a comprehensive analysis at 6, 10, 14, 18 and 20 DAA, integrating morphological, histological, and transcriptomic approaches.

Results and discussion: Morphological and histochemical data revealed that by 10 DAA, cotyledons are fully formed, but storage compound accumulation is only noticed at 14 DAA, indicating that the transition from embryogenesis to seed filling occurs between 10 and 14 DAA. Transcriptomic analysis further supported this finding, showing upregulation of genes associated with seed storage proteins, starch metabolism, and hormonal regulation at 14 and 18 DAA. This study redefines the developmental timeline of P. vulgaris seed filling initiation, bridging a critical knowledge gap in legume seed biology. Given the limited availability of histological studies on early P. vulgaris seed development, our findings provide essential insights into the structural and molecular events driving this transition. By refining the timing and regulatory mechanisms of early seed development, this study lays the groundwork for future research aimed at enhancing seed quality and resilience in legumes.

Keywords: Phaseolus vulgaris L.; early seed filling; seed histology; storage compounds; transcriptome.

Figure 1

Characterization of seed development in P. vulgaris SER16 at 6, 10, 14, 18 and 20 DAA (days after anthesis). The study was focused on the transition from seed embryogenesis to seed filling. (a) transversal section of seeds at 6, 10, 14, 18 and 20 DAA at the Hilum level, black scale bars indicate 1 mm; (b) Photographs of seeds at 6, 10, 14, 18 and 20 DAA; (c) Seed fresh weight (SFW), Seed dry weight (SDW), Seed length (SL). Error bars represent the standard deviation and different letters indicate statistically significant differences between time points (p<0.05).

Figure 2

Morphology and histology of developing P. vulgaris SER16 seeds harvested at 6, 10, 14, 18 and 20 days after anthesis (DAA). Histological observations of: (a) cell walls, (b) protein and (c) starch accumulation during seed development. The cell walls, proteins and starch were stained with Calcofluor white stain, Coomassie Brilliant Blue and Periodic Acid Schiff, respectively. The white scale bars indicate 124.4 µm and black scale bars indicate 248.9µm; abpy - cotyledon abaxial parenchyma; adpy - cotyledon adaxial parenchyma; C, cotyledon; cw, cell wall; doc, dermal cell complex; e, embryo; p, parenchyma; psv, protein storage vacuoles; SC, seed coat; sg, starch grains; vb, vascular bundle.

Figure 3

Changes in cotyledon parenchyma cell number and area of P. vulgaris SER16 seeds, from 10 to 20 DAA (days after anthesis). (a) Cotyledon parenchyma cell mean area. (b) Cotyledon transversal section mean area. Mean gray value for pixel intensity (obtained using Image J software after converted to a grayscale) for: (c) Coomassie blue and (d) Periodic acid Schiff. Different letters above each bar indicates statistically significant differences (p < 0.05).

Figure 4

Functional categories of differentially expressed genes (DEGs) in the transition from the embryogenesis to seed filling in P. vulgaris SER16 seeds. The percentage of DEGs in each category were displayed between the main comparison studied (6 DAA vs 10 DAA; 10 DAA vs 14 DAA and 14 DAA vs 18 DAA). The percentage of DEGs changed was calculated by comparison of the number of DEGs in each category in relation to the total of DEGs identified within each comparison.

Figure 5

Seed storage protein gene expression. (a) Domain structure of Phaseolin exhibiting the bi-cupin architecture (Emani and Hall, 2008); (b) Heatmap of transcripts related to seed storage proteins at 6, 10, 14 and 18 days after anthesis (DAA). FPKM were clustered using Euclidean distance and an average linkage. Blue color represents low expression levels (lower FPKM values); White (Middle Color) represents moderate expression levels and Red represents high expression levels (higher FPKM values). Asterisks indicate differentially expressed genes (DEGs): corrected p‐value ≤ 0.001 and |Log2(Fold Change - FC)≥1.

Figure 6

Heatmaps of transcripts of P. vulgaris SER16 seeds, categorized in MapMan, related to sucrose and starch synthesis at 6, 10, 14 and 18 days after anthesis (DAA). Fructose 1–6 biphosphatase (FBP1), Phosphoglucose isomerase, Phosphoglumatase (PGM), Sucrose phosphate synthase (SPS), Sucrose phosphate phosphatase (SPP), UDP-glucose pyrophosphorylase (UDP), ADP-glucose pyrophosphorylase (AGPase), Plastid starch phosphorylase (Pho1), Starch synthase (SS), Granule-bound starch synthase (GBSS), Starch branching enzyme (SBE), Isoamylases (ISA), Pullulanase (PUL), α-amylase and β-amylase. Schematic representation of sucrose and starch synthesis adapted from (Qu et al., 2018). FPKM were clustered using Euclidean distance and an average linkage. Blue color represents low expression levels (lower FPKM values); White (Middle Color) represents moderate expression levels and Red represents high expression levels (higher FPKM values). Asterisks indicate differentially expressed genes (DEGs): corrected p‐value ≤ 0.001 and |Log2(Fold Change - FC)≥1.

Figure 7

Heatmaps of transcripts of P. vulgaris SER16 seeds, categorized in MapMan, related to LAFL network genes found differentially expressed between 6, 10, 14 and 18 days after anthesis (DAA). LAFL activation genes are shown in purple, LAFL repression genes in pink and LAFL direct target genes in blue. The yellow box highlight the major genes of the LAFL network (LEC1, L1L, ABI3, FUS3 and LEC2) and illustrates their regulatory interactions. Schematic representation adapted from (Devic and Roscoe, 2016; Jia et al., 2014). FPKM were clustered using Euclidean distance and an average linkage. Blue color represents low expression levels (lower FPKM values); White (Middle Color) represents moderate expression levels and Red represents high expression levels (higher FPKM values). Asterisks indicate differentially expressed genes (DEGs): corrected p‐value ≤ 0.001 and |Log2(Fold Change - FC)≥1.

Figure 8

Heatmaps of transcripts of P. vulgaris SER16 seeds, categorized in MapMan, related to plant growth regulator pathways, including key components of the abscisic acid (ABA), jasmonic acid (JA), cytokinin (CK), gibberellin (GA), ethylene (ET), auxin (AUX) and brassinosteroid (BR) pathways, which are regulated by the LAFL network, found differentially expressed between 6, 10, 14 and 18 days after anthesis (DAA). FPKM were clustered using Euclidean distance and an average linkage. Blue color represents low expression levels (lower FPKM values); White (Middle Color) represents moderate expression levels and Red represents high expression levels (higher FPKM values). Asterisks indicate differentially expressed genes (DEGs): p‐value ≤ 0.001 and |Log2(Fold Change - FC)≥1.