**5. Priming and seed water content**

Seed germination is characterized by triphasic kinetic of water uptake with a rapid initial uptake (imbibition), followed by an apparent lag phase. A further increase in water uptake occurs only after germination is completed, as the embryonic axis elongates [111] (**Figures 1** and **2**). Early imbibition of seeds involves the fastest and most drastic changes in tissue hydration observed during germination. The water content of seeds and tissues within seeds depends of the composition of stored reserves. Seed imbibition and subsequent embryo growth depend on water exchanges and water potential gradients represent the motive force for water flow and finally tissue expansion. Water transport across cell membranes is essential for the initiation of metabolism. This intracellular water transport is mediated by aquaporins.

Aquaporins (AQPs) are transmembrane proteins, members of the major intrinsic protein (MIP) family that facilitate rapid and passive water transport across cell membranes and play a crucial role in plant water relations [112–114]. Plant aquaporins are remarkably diverse with several subfamilies of MIPs identified in dicots and monocots. Among them, the plasma membrane intrinsic proteins (PIPs) and the tonoplast intrinsic proteins (TIPs) subfamilies constitute the largest number of AQPs and correspond to AQPs that are abundantly expressed in the plasma and vacuolar membranes, respectively. Both PIP and TIP subfamilies are believed to play a key role in transcellular and intracellular plant water transport.

To gain insight into the role of water channel in germination, the expression profiles of *AQP* genes were studied in *Arabidopsis* [115], *Oryza sativa* [116], and *Brassica napus* [117] during seed imbibition and early embryo growth. These results have demonstrated the possible role of several AQPs in seed germination also in response to abiotic stresses. Moreover, Liu et al. [116] have shown the reduced seed germination rate via *OsPIP1;3* silencing and the promotion of seed germination via *OsPIP1;3* over-expression under drought conditions demonstrating that OsPIP1;3 is required for normal seed germination.

Seed priming involves imbibing seeds with restricted amounts of water to allow hydration sufficient to permit pre-germinative metabolic events to proceed while preventing radicle protrusion. This treatment can extend phase II of water uptake while preventing seeds from entering into phase III. The completion of radicle emergence is prevented by restricted amount of water provided to the seed (hydropriming, solid matrix priming) or decreased water potential (*Ψ*w) of the imbibition medium by the use of osmotic solutes such as PEG or salts (osmopriming) [111]. In a study on *Brassica napus* osmopriming, Kubala et al. [36] revealed that at the beginning of the soaking period and at the end of drying phase, the seed water content was as low as 5%. The soaking treatment allowed seed imbibitions up to 50%, which should be enough to re-initiate metabolism.

As AQPs regulate water movement, it can be supposed that these proteins play an important role both in priming treatment (seed soaking) and post-priming germination under both favorable and unfavorable conditions. A role for aquaporin-controlled water transport across cell membranes in primed seeds of *Brassica napus* during germination was demonstrated by Gao et al. [118]. Seed priming with PEG or ABA resulted in an enhanced germination, particularly under salt and osmotic stresses at low temperature. Priming treatment induced expression of *BnPIP1* but had no effect on transcript level of Bnγ*-TIP2*. However, transcripts of both Bn*PIP1* and Bnγ*-TIP2* genes during germination were present earlier in primed seeds than nonprimed ones. Gao et al. [118] speculated that Bn*PIP1* was involved in water transport required for the activation of enzymatic metabolism of storage nutrients in the early stages of rapeseed germination, while Bnγ*-TIP2* expression was correlated with cell growth during radicle emergence. Changes in the expression pattern of *SoPIP1;1, SoPIP1;2, SoPIP2;1*, and *SoδTIP* during *Spinacia oleracea* seeds osmopriming and post-priming germination under optimal conditions, chilling and drought have been reported by Chen et al. [119]. The authors have stated that all these genes were up-regulated within 2–4 d of priming (phase II-imbibi‐ tion). Therefore, the high expression of those *AQPs* might contribute to water transport across plasma and vacuolar membranes to facilitate water supply to expanding tissues and to increase germination potential of primed seeds. The down-regulation of all *AQP*s genes expression was observed under chilling and drought. However, the expression of some *AQPs* genes was elevated in primed seeds that also exhibited greater chilling and drought tolerance [119]. Kubala et al. [36] revealed up-regulation of two genes encoding tonoplast AQPs (*TIP4.1* and *TIP1.2*) in *Brassica napus* seeds in relation to osmopriming. In this study, expression of *TIP1.2* increased approximately 20 fold during post-priming germination as compared to unprimed seeds. In addition, the same authors have also stated facilitated water uptake and higher stress tolerance of germinating primed *Brassica napus* seeds [26, 120]. The above-mentioned results have demonstrated that water transport and sufficient water supply for embryo during postpriming germination regulated by AQPs may be one of the crucial components modulated by pre-sowing seed priming that influences germination rate and stress resistance.

**5. Priming and seed water content**

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

OsPIP1;3 is required for normal seed germination.

be enough to re-initiate metabolism.

Seed germination is characterized by triphasic kinetic of water uptake with a rapid initial uptake (imbibition), followed by an apparent lag phase. A further increase in water uptake occurs only after germination is completed, as the embryonic axis elongates [111] (**Figures 1** and **2**). Early imbibition of seeds involves the fastest and most drastic changes in tissue hydration observed during germination. The water content of seeds and tissues within seeds depends of the composition of stored reserves. Seed imbibition and subsequent embryo growth depend on water exchanges and water potential gradients represent the motive force for water flow and finally tissue expansion. Water transport across cell membranes is essential for the initiation of metabolism. This intracellular water transport is mediated by aquaporins.

Aquaporins (AQPs) are transmembrane proteins, members of the major intrinsic protein (MIP) family that facilitate rapid and passive water transport across cell membranes and play a crucial role in plant water relations [112–114]. Plant aquaporins are remarkably diverse with several subfamilies of MIPs identified in dicots and monocots. Among them, the plasma membrane intrinsic proteins (PIPs) and the tonoplast intrinsic proteins (TIPs) subfamilies constitute the largest number of AQPs and correspond to AQPs that are abundantly expressed in the plasma and vacuolar membranes, respectively. Both PIP and TIP subfamilies are

To gain insight into the role of water channel in germination, the expression profiles of *AQP* genes were studied in *Arabidopsis* [115], *Oryza sativa* [116], and *Brassica napus* [117] during seed imbibition and early embryo growth. These results have demonstrated the possible role of several AQPs in seed germination also in response to abiotic stresses. Moreover, Liu et al. [116] have shown the reduced seed germination rate via *OsPIP1;3* silencing and the promotion of seed germination via *OsPIP1;3* over-expression under drought conditions demonstrating that

Seed priming involves imbibing seeds with restricted amounts of water to allow hydration sufficient to permit pre-germinative metabolic events to proceed while preventing radicle protrusion. This treatment can extend phase II of water uptake while preventing seeds from entering into phase III. The completion of radicle emergence is prevented by restricted amount of water provided to the seed (hydropriming, solid matrix priming) or decreased water potential (*Ψ*w) of the imbibition medium by the use of osmotic solutes such as PEG or salts (osmopriming) [111]. In a study on *Brassica napus* osmopriming, Kubala et al. [36] revealed that at the beginning of the soaking period and at the end of drying phase, the seed water content was as low as 5%. The soaking treatment allowed seed imbibitions up to 50%, which should

As AQPs regulate water movement, it can be supposed that these proteins play an important role both in priming treatment (seed soaking) and post-priming germination under both favorable and unfavorable conditions. A role for aquaporin-controlled water transport across cell membranes in primed seeds of *Brassica napus* during germination was demonstrated by Gao et al. [118]. Seed priming with PEG or ABA resulted in an enhanced germination,

believed to play a key role in transcellular and intracellular plant water transport.

As PIPs, but not TIP, are generally found at the plasma membrane, PIPs are thought to play a key role in seed water uptake. Nevertheless, both microarray [121] and macroarray experi‐ ments [115] with the complete set of genes encoding major intrinsic proteins revealed that out of 13 PIPs encoded by the *Arabidopsis* genome, transcripts for only three isoforms (PIP1;2, PIP1;4, and PIP1;5) were detectable in seeds. Mapping of TIPs in germinating *Arabidopsis* seeds has revealed that isoforms TIP3;1 and TIP3;2 detected in embryos, appear to localize to both the plasma membrane and tonoplast [122]. Vander Willigen et al. [115] have observed that during germination, very high level of TIP3 protein was coincident with decreased level of PIP1;2 and PIP2;1 polypeptides until phase III of water uptake. As stated by Vander Willigen et al. [115], it is intriguing how such low concentrations of PIP protein during the early phases of germination can achieve basic transcellular water transport in the seed. Gattolin et al. [122] have speculated that TIP3 may be the only AQP involved in seed water intake, and that the presence of TIP3 at the plasma membrane may compensate for the absence (or low concen‐ tration) of PIPs. In the light of these results, the enhanced germination potential of primed *Brassica napus* seeds could be partially explained by the up-regulation of TIPs during priming and post-priming germination [36]. However, the involvement of apoplastic water movement and simple diffusion of water across membranes during seed imbibition cannot be ruled out. The treatment of *Arabidopsis* seeds with mercury, a general blocker of aquaporins, reduced the speed of seed germination but did not affect its developmental sequence or basic aspects of seed water relations. Vander Willigen et al. [115] suggested that aquaporins functions are not involved in early seed imbibition but would rather be associated with water uptake accom‐ panying expansion and growth of the embryo.

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 in seeds.

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 contribute to rapid seed germination by improving water uptake.

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

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 proteins and the loss of membranes integrity. This could be realized due to the ability of most LEA proteins to either coat intracellular macromolecules with a coherent layer of water or to interact with the surface of proteins and thus acting as water replacement [135]. As LEA proteins accumulate at a high level in response to cell/tissue dehydration, they may contribute to acquisition of tolerance to drought and related stresses such as osmotic, salt, and cold stress. In support of this, several studies revealed changes in the pattern of expression/accumulation of LEA transcript/protein in seeds caused by priming treatment and suggested their associa‐ tion with improved stress tolerance of primed seeds [36, 107, 136–138]. For example, the transcripts of two genes: *Em6*, encoding LEA group 1 protein and *RAB18*, encoding responsive to ABA 18 protein, belonging to LEA group 2, declined during osmopriming (soaking in PEG solution), reaccumulated after slow drying and again degraded during *Brassica oleracea* seeds germination [138]. The up-regulation of *RAB18* and *Em6* expression during slow seed drying suggests that they play a role in drought tolerance. Chen et al. [107] reported transient accumulation of four dehydrins-like proteins (32, 30, 26, 19-kD) in seeds of *Spinacia oleracea* during early stages of osmopriming followed by progressive degradation to a lower level in primed dry seeds compared to unprimed ones. A similar trend was confirmed to cold acclimation protein CAP85. In contrast to protein concentration, relative expression of *CAP85* was greater in primed dry seeds than in unprimed ones. Recently, Kubala et al. [36] revealed accumulation of LEA transcripts (*LEA4-1, LEA4-5*) and LEA3 proteins during soaking in PEG solution. The authors proposed that soaking in PEG with a low osmotic potential should not be considered only as a rehydration phase: water uptake may be sufficient to reinitiate a physiological activity from a previous quiescent stage but water content of 50% remained low enough to represent a water stress situation, especially when it is maintained during several days [36].
