Unveiling the secrets of lotus seed longevity: insights into adaptive strategies for extended storage

Abstract

Seed longevity is crucial for long-term storage, but prolonged unfavorable conditions can lead to loss of viability. This study integrated theoretical and experimental techniques to elucidate the inherent mechanisms underlying the unique ability of lotus seeds to maintain stable viability over many years. Transcriptome analysis and microscopy revealed a sturdy structure of the lotus seed pericarp, which predominantly expressed cellulose synthase genes involved in cell wall biogenesis. The cotyledon serves as a nutrient source for seeds during long-term storage. Additionally, the inactivation of chlorophyll degradation pathways may allow for the retention of chlorophyll in the lotus seed plumule, potentially enhancing the environmental adaptability of lotus seedlings. Reduced abundance of transcripts corresponding to heat shock protein genes could impact protein processing and consequently diminish the vitality of aging lotus seeds. Moreover, an expansion in the number of seed maturation and defense response genes was observed in the lotus genome compared with 11 other species, which might represent an adaptive strategy against long-term adverse storage conditions. Overall, these findings are crucial for understanding the mechanisms underlying lotus seed longevity and may inform future improvements in the extended storage periods of seed crops.

Keywords: Ancient lotus seed; accelerated aging; genome; longevity; lotus; transcriptome.

Fig. 1.

Lotus seed pericarp observed at 15 and 30 days after pollination (DAP). (A) The lotus seed structure. Scale bar: 1 cm. (B, C) Light microscopy (B) and scanning electron microscopy (C) images of lotus seed pericarp. C, cavity; EC, epidermal cell; Pa, parenchymal cell; PC, palisade cell; SC, secretory cell. (D) Contents of total flavonoids and total phenolics in the seeds of the lotus cultivar ‘Jianxuan17’. (E) Antioxidant activities in the seeds of ‘Jianxuan17’. ABTS, 2,2ʹ-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid); DPPH, 2,2-diphenyl-1-picrylhydrazyl; FRAP, ferric ion reducing antioxidant potential. Values represent means ±SE (n=3). Statistical significance is based on one-way ANOVA with Tukey’s test at P<0.05. Different letters over the bars indicate significant difference.

Fig. 2.

RNA-seq analysis of seed pericarp of the lotus cultivar ‘Jianxuan17’. (A) Morphological changes in lotus seed pericarp during development. DAP, days after pollination. (B) The number of up- and down-regulated genes in different comparison groups. (C) Venn diagram showing overlapping differentially expressed genes (DEGs) in different comparison groups. (D) Gene ontology (GO) functional enrichment of up- (left) and down-regulated (right) genes in different comparison groups. Each comparison group showed only the top five pathways. (E) Co-expressed modules from DEGs based on weighted gene co-expression network analysis. (F) GO functional enrichment of genes in the yellow co-expression module shown in (E). The red text highlights several biological processes associated with lotus seed pericarp morphogenesis, such as cell wall thickening and plant-type primary/secondary cell wall biogenesis.

Fig. 3.

Identification of NnCesA genes involved in cell wall biosynthesis during lotus seed pericarp development. (A) Schematic representation of lotus seed cell wall structure. (B) Contents of cellulose, hemicellulose, and lignin in the 30 days after pollination (DAP) seed pericarp of the lotus cultivar ‘Jianxuan17’. Values represent means ±SE (n=3). Statistical significance is based on one-way ANOVA with Tukey’s test at P<0.05. Different letters over the bars indicate significant difference. (C, D) Heatmaps of the plant-type primary (C) and secondary (D) cell wall biosynthesis gene expression patterns. NnCesA gene IDs are shown in red. (E) Phylogenetic tree showing the relationship of NnCesA genes with other plants. (F) qRT-PCR validation of six NnCesA genes. Values represent means ±SE (n=3). NnActin was used as the internal control.

Fig. 4.

Biosynthesis of starch in lotus seed cotyledon. (A) Structural composition of the major tissues at 30 days after pollination (DAP) of seeds of the lotus cultivar ‘Jianxuan17’. (B) The contents of starch, protein, and soluble sugar in the cotyledons of 30 DAP seeds of ‘Jianxuan17’. (C) Starch accumulation in the seed cotyledons of ‘Jianxuan17’ at different developmental stages. Values represent means ±SE (n=3). Statistical significance is based on one-way ANOVA with Tukey’s test at P<0.05. Different letters over bars indicate significant difference. (D) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of 3066 differentially expressed genes. (E) Heatmap showing the expression patterns of starch and sucrose metabolic pathway genes. CA, ‘China Antique’; JX, ‘Jianxuan17’.

Fig. 5.

Stress response and chlorophyll retention during lotus seed plumule development. (A) Co-expressed modules from 16 905 differentially expressed genes (DEGs) in lotus seed plumule based on WGCNA. (B) GO functional enrichment of genes in the blue co-expression module. The red text indicates the pathways associated with responses to water deficit, cold, heat, and hypoxia. (C) Visualization of WGCNA for co-expressed genes in the blue co-expression module. The genes in the green, pink, purple, and orange circles are associated with water deficit response, cellular response to hypoxia, abscisic acid, and heat response, respectively. (D) Chlorophyll biosynthesis and degradation pathways in lotus seed plumule. Pchlide, protochlorophyllide. (E) Heatmap showing the expression of chlorophyll biosynthesis and degradation genes during lotus seed plumule development.

Fig. 6.

Transcriptome analysis of natural aging lotus seeds. (A) Phenotypes of the ancient lotus seeds and their progeny (modern seeds). Scale bar: 1 cm. (B) Contents of cellulose, hemicellulose, and lignin. (C) Content of total flavonoids. (D) Detection of antioxidant activities in the pericarp of ancient and modern lotus seeds. ABTS, 2,2ʹ-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid); FRAP, ferric ion reducing antioxidant potential. Values represent means ±SE (n=3). **P≤0.01, t-test. (E, F) KEGG analysis of up- and down-regulated genes (E) and GO analysis of up- and down-regulated genes (F) in the plumule of the modern lotus seeds. (G) Heatmap showing the expression of differentially expressed heat shock protein (HSP) genes in natural and accelerated aging experiments. DEG, differentially expressed gene. (H) qRT-PCR validation of six NnHSP genes. Values represent means ±SE (n = 3). NnActin as an internal control.

Fig. 7.

The expanded genes in the lotus genome. (A) The number of expanded orthogroups in the lotus genome. (B) GO functional enrichment of expanded genes in lotus. (C) Variation in the number of polygalacturonase inhibitor protein (PGIP) genes in different plant species. (D) Phylogenetic tree of NnPGIP genes. (E) Chromosomal localization of NnPGIP genes.