**6. Priming and seed ultrastructure**

involved in early seed imbibition but would rather be associated with water uptake accom‐

The transmembrane water transport via the regulation of AQP quantity and activity endows seeds with a remarkable capacity to modulate water absorption, transport, and compartmen‐ tation within tissues. Nuclear magnetic resonance (NMR) spectroscopy has provided insights into changes in the physical states of seed water during germination [123–126]. In particular, magnetic resonance imaging (MRI) has revealed a precise spatial distribution of water within tissues of germinating seeds and different patterns between species [125, 127, 128] highlighting the tight control of water transport. Water status of primed seeds was characterized by Nagarajan et al. [129] in study on tomato halo-and osmopriming. Nagarajan et al. [129] pointed out that better performance of primed seeds may be attributed to the modifications of seed water-binding properties and reorganization of seed water during imbibition, so as to increase the macromolecular hydration water required for various metabolic activities related to the germination process. In the future, it will be crucial to see how the spatial pattern of aquaporin expression can fit the hydration pattern revealed by MRI during both priming and postpriming germination, therefore enabling a comprehensive understanding of water transport

Several studies have reported that water uptake is improved by priming treatment as primed seeds exhibited a faster imbibition in comparison with nonprimed ones, although pre-treated seeds were dried after priming to reach the same water content as nonprimed ones [36, 47, 107]. Although MRI studies revealed that water penetrates seeds through the hilum and micropyle [125, 130], Galhaut et al. [47] did not observe any particular modification of these structures after *Trifolium repens* priming, despite a faster seed hydration. However, scanning electron microscopy analysis showed that primed seeds of *Trifolium repens* exhibited seed coat tears and circular depressions that can favor seed imbibition. Moreover, X-ray photographs revealed tissue detachment in dry primed seeds that formed free space between the cotyledons and radicle, making water flow easier, thus contributing to tissue hydration [47]. Similarly, formation of free space surrounding the embryo in dry primed seeds of tomato was noticed by Liu et al. [131]. In brief, these observations suggest that structural modifications might

The maintenance of favorable water status is critical for survival of germinating seeds under environmental stresses leading to tissue dehydration. The accumulation of nontoxic, compat‐ ible solutes within seed tissues, that is, osmotic adjustment is a major trait associated with maintenance of high cell turgor pressure potential in response to stress conditions. Priming treatment itself may generate a moderate abiotic stress during soaking (e.g. osmotic stress, salt, and drought created by the priming agents) [36, 132]. The accumulation of osmotically active solutes such as amino acids (e.g. proline) ammonium compounds (e.g. glycine betaine), sugars (e.g. glucose, fructose, sucrose) during priming was noticed in several species and was shown

Seeds can also experience dehydration in the course of priming treatment, that is, during drying after soaking. Late embryogenesis abundant proteins (LEAs) can stabilize cell structure and macromolecules upon cell dehydration by preventing inactivation and aggregation of

contribute to rapid seed germination by improving water uptake.

to improve seed germination under subsequent water stress [3, 21, 133, 134].

panying expansion and growth of the embryo.

16 New Challenges in Seed Biology - Basic and Translational Research Driving Seed Technology

in seeds.

In general, the ability of seeds to germinate seems to be critically determined by a change in the balance between growth potential of the embryo and the mechanical resistance of the surrounding tissues. In many species, the endosperm tissue enclosing the embryo restrains the germination process acting as a physical barrier, which restricts radicle emergence.

Weakening of tissues surrounding elongating radicle by cell separation due, for instance, to the activity of cell wall hydrolases may occur as a consequence of priming (**Figure 2**). It was determined that osmopriming induced hydrolysis of the endosperm tissue of *Cucumis melo* seeds [139] and increased the endo-β-mannanase activity in the endosperm cap and decreased its mechanical restraint on the elongating tomato embryo [140]. A strong correlation was observed between lowering of the mechanical restraint and the activity of endo-β-mannanase [141].

Penetration of the structures surrounding the embryo is a consequence of radicle cells elongation. Up-regulation of the gene encoding xyloglucan endotransglucosylase/hydrolase (XTH) in response to osmopriming and accumulation of transcript for extensin-like protein (ELP) during post-priming germination was observed in rapeseed [36]. As XTHs have the ability to cleave xyloglucans and rejoin the cut ends with new partners, they are engaged in cell wall loosening during growth and in the restructuring of the cell walls after extension.

Cytoskeleton reorganization is also necessary to achieve large rates of cells elongation that precedes radicle protrusion. The component of microtubules (β-tubulin) accumulated in tomato seeds during germination and priming and the expression preceded visible germina‐ tion [142]. Higher level of β-tubulin protein accumulation was shown in rapeseed during PEG soaking, drying, and post-priming germination. The up-regulation of genes encoding γ- and β-tubulins was also noticed during post-priming germination [36].

Ultrastructural observations performed during the 6-d period of solid matrix priming (SMP) of carrot (*Daucus carrota*) seeds indicated the breakdown of storage materials, specific to the catabolic phase of germination *sensu stricto*, both in the axis and in the micropylar endosperm covering the radicle tip [143]. It was found that complete degradation of storage protein and lipid bodies and subsequent starch accumulation occurred in the radicles of carrot seeds after 8-d SMP. In the endosperm, the catabolic changes were limited to the micropylar area, where extensive breakdown of storage cell walls, partial degradation of protein bodies, and no storage lipid hydrolysis were observed [144].

During seed germination, storage proteins, which provide a source of reduced nitrogen, and inorganic minerals need to be mobilized to support seedling growth. In addition, a lytic aqueous vacuolar compartment building up the turgescence necessary for cell expansion and to promote radicle protrusion and embryo elongation has to be formed (**Figure 2**). Bolte et al. [145] investigated the features and the dynamics of the vacuoles during the early stages of *Arabidopsis* seed germination and indicated the successive occurrence of two different lytic compartments in the protein storage vacuoles (PSV). The first one corresponds to globoids specialized in mineral storage and the second one is at the origin of the central lytic vacuole in these cells [145]. Different mechanisms for the transformation of PSV into lytic vacuole in the root tip cell of germinating tobacco (*Nicotiana tabacum*) seeds were proposed by Zheng and Staehelin [146]. Ultrastructural studies demonstrated that the radicle cells of tobacco contain only one type of vacuole at particular time of development. Upon rehydration, the radicle cells only contain PSVs, but during subsequent root development, the PSVs are systematically transformed into lytic vacuoles via cell type specific pathways.

At present, we do not have a complete view of ultrastructural changes occurring during seed priming. One would expect that similar vacuole remodeling might occur during priming, especially in embryonic axis. The maintenance of metabolism over, for instance, several days of seed priming requires mobilization of embryo reserves. It is speculated that accumulation of endogenous osmotica, cell vacuolization, together with cell wall loosening initiated during priming, might determine the embryonic axis extension and radicle protrusion during postpriming germination. Deeper and more detailed studies should be continued in order to completely clarify this phenomenon.
