The origin of the agriculture was one of the turning points in human history, and a central part of this was the evolution of new plant forms, domesticated crops. Seed dispersal and germination are two key traits which have been selected to facilitate cultivation and harvesting of crops. The objective of this study was to analyze anatomical structure of seed coat and pod, identify metabolic compounds associated with water-impermeable seed coat and differentially expressed genes involved in pea seed dormancy and pod dehiscence. Comparative anatomical, metabolomics, and transcriptomic analyses were carried out on wild dormant, dehiscent Pisum elatius (JI64, VIR320) and cultivated, indehiscent Pisum sativum non-dormant (JI92, Cameor) and recombinant inbred lines (RILs). Considerable differences were found in texture of testa surface, length of macrosclereids, and seed coat thickness. Histochemical and biochemical analyses indicated genotype related variation in composition and heterogeneity of seed coat cell walls within macrosclereids. Liquid chromatography–electrospray ionization/mass spectrometry and Laser desorption/ionization–mass spectrometry of separated seed coats revealed significantly higher contents of proanthocyanidins (dimer and trimer of gallocatechin), quercetin, and myricetin rhamnosides and hydroxylated fatty acids in dormant compared to non-dormant genotypes. Bulk Segregant Analysis coupled to high throughput RNA sequencing resulted in identification of 770 and 148 differentially expressed genes between dormant and non-dormant seeds or dehiscent and indehiscent pods, respectively. The expression of 14 selected dormancy-related genes was studied by qRT-PCR. Of these, expression pattern of four genes: porin (MACE-S082), peroxisomal membrane PEX14-like protein (MACE-S108), 4-coumarate CoA ligase (MACE-S131), and UDP-glucosyl transferase (MACE-S139) was in agreement in all four genotypes with Massive analysis of cDNA Ends (MACE) data. In case of pod dehiscence, the analysis of two candidate genes (SHATTERING and SHATTERPROOF) and three out of 20 MACE identified genes (MACE-P004, MACE-P013, MACE-P015) showed down-expression in dorsal and ventral pod suture of indehiscent genotypes. Moreover, MACE-P015, the homolog of peptidoglycan-binding domain or proline-rich extensin-like protein mapped correctly to predicted Dpo1 locus on PsLGIII. This integrated analysis of the seed coat in wild and cultivated pea provides new insight as well as raises new questions associated with domestication and seed dormancy and pod dehiscence.

Seed coats of six pea genotypes contrasting in dormancy were studied by laser desorption/ionization mass spectrometry (LDI-MS). Multivariate statistical analysis discriminated dormant and non-dormant seeds in mature dry state. Separation between dormant and non-dormant types was observed despite important markers of particular dormant genotypes differ from each other. Normalized signals of long-chain hydroxylated fatty acids (HLFA) in dormant JI64 genotype seed coats were significantly higher than in other genotypes. These compounds seem to be important markers likely influencing JI64 seed imbibition and germination. HLFA importance was supported by study of recombinant inbred lines (JI64xJI92) contrasting in dormancy but similar in other seed properties. Furthemore HLFA distribution in seed coat was studied by mass spectrometry imaging. HLFA contents in strophiole and hilum are significantly lower compared to other parts indicating their role in water uptake. Results from LDI-MS experiments are useful in understanding (physical) dormancy (first phases of germination) mechanism and properties related to food processing technologies (e.g., seed treatment by cooking).

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