Recently developed studies of aquaporins, the proteins forming water channels in animal membranes, culminated in the Nobel Prize ‘For the discovery of water channels’ awarded to Peter Agre, an American scientist, in 2003. This new field of science has attracted the attention of plant physiologists dealing with the mechanisms of water transport. Previously, water transport was believed to occur by slow diffusion, and the possibility of the functioning of special water channels has been of obvious interest. A number of reviews concerning aquaporins have appeared recently (Maurel, Reference Maurel1997; Tyerman et al., Reference Tyerman, Bohnert, Maurel, Steudle and Smith1999; Chaumont et al., Reference Chaumont, Barrieu, Wojcic, Chrispeels and Jung2001; Maurel et al., Reference Maurel, Verdoucq, Luu and Santoni2008, Reference Maurel, Santoni, Luu, Wudick and Verdoucq2009) demonstrating rapid penetration of water through water channels in the course of transcellular osmotic-driven movement. Water influx into the plant cell occurs through water channels formed by plasmalemma aquaporins (PIPs), whereas the delivery of excessive water to the vacuole depends on tonoplast aquaporins (TIPs). [According to universally accepted nomenclature, the PIP subfamily embraces plasmalemma intrinsic proteins, the TIP subfamily consists of tonoplast intrinsic proteins and the NIP subfamily includes nodulin-like intrinsic proteins. The first number indicates subtype, the second one indicates the number of the isoform. Previous names of TIPs were αTIP (TIP3;1), γTIP (TIP1) and δTIP (TIP2).] The rate of water transport along the channels exceeds the rate of water diffusion by at least tenfold. The efficiency of water transport depends on aquaporin content in the membranes and an opened or closed state of the channels, which is mainly regulated by phosphorylation/dephosphorylation, respectively (Maurel et al., Reference Maurel, Verdoucq, Luu and Santoni2008).

Over the course of seed development, the earliest appearance of PIP2 gene transcripts was shown in the ovules of Solanum chacoense at 48–60 h after pollination, i.e. just after fertilization (O'Brien et al., Reference O'Brien, Bertrand and Matton2002). During embryogenesis of Pinus taeda seeds, the expression of the NIP1;1 gene was reported in the suspensors of both zygotic and somatic embryos (Ciavatta et al., Reference Ciavatta, Morillon, Pullman, Chrispeels and Cairney2001). The NIP aquaporin belongs to the aqua-glyceroporins that are capable of transporting both water and glycerol.

In developing pea seeds, the expression of PIP1;1, PIP2;1 and TIP1;1 genes occurred in expanding cotyledons and in the seed coat, where expression of NIP1;1 was also observed (Schuurmans et al., Reference Schuurmans, van Dongen, Rutjens, Boonman, Pieterse and Borstlap2003). PIP2;1 participates in water transport to a greater extent than NIP1;1 and TIP1;1. In the experiments of Zhou et al. (Reference Zhou, Setz, Niemietz, Qu, Offler, Tyerman and Patrick2007) with developing Phaseolus vulgaris seeds, an active expression of PIP1;1, PIP2;2 and PIP2;3 genes, as well as their water-channel activities, were shown in vascular parenchyma of seed-coat bundles, where water unloading from the phloem to the apoplast occurs. These data indicate participation of the water channel in the delivery of water, and possibly some organic compounds, into the developing seeds.

Much more attention was paid to tonoplast aquaporins in the course of vacuole transformation to protein storage vacuoles in maturing orthodox seeds. TIP3;1 is considered a marker of the tonoplast (Johnson et al., Reference Johnson, Herman and Chrispeels1989; Jauh et al., Reference Jauh, Philips and Rogers1999; Hunter et al., Reference Hunter, Craddock, Benedetto, Roberts and Frigerio2007). It is specifically produced in protein-storing tissues of monocots, dicots and gymnosperms (Melroy and Herman, Reference Melroy and Herman1991; Höfte et al., Reference Höfte, Habbard, Reizer, Ludevid, Herman and Chrispeels1992; Inoue et al., Reference Inoue, Takeuchi, Nishimura and Hara-Nishimura1995; Oliviusson and Hakman, Reference Oliviusson and Hakman1995; Herman and Larkins, Reference Herman and Larkins1999; Takahashi et al., Reference Takahashi, Rai, Kitagawa, Morita and Masumura2004; Willigen et al., Reference Willigen, Postaire, Tournaire-Roux, Boursiac and Maurel2006; Li et al., Reference Li, Peng, Yu, Zhang, Cai, Sun and Su2008). In the case of recalcitrant seeds, for example Aesculus hippocastanum, TIP3;1 proteins remained in the tonoplast of preserved vacuoles during maturation, shedding and germination (Obroucheva et al., Reference Obroucheva, Lityagina, Novikova and Sinkevich2012). However, in maturing orthodox seeds, the vacuoles are transformed to protein-storage vacuoles as a result of the deposition of reserve proteins therein. TIP3;1 accumulated there when most of the storage proteins had already been delivered (Johnson et al., Reference Johnson, Herman and Chrispeels1989; Melroy and Herman, Reference Melroy and Herman1991). TIP3;1 proteins are synthesized in maturing seeds by the ribosomes on the endoplasmic reticulum and then transported in Golgi vesicles to the vacuole (Mäder and Chrispeels, Reference Mäder and Chrispeels1984). This process was studied in more detail in pumpkin cotyledons (Inoue et al., Reference Inoue, Takeuchi, Nishimura and Hara-Nishimura1995). mRNA of the TIP3;1 precursor was detected at early cotyledon development, and this precursor, of size 29 kDa, gradually accumulated. Its conversion to the end product occurred by removal of the terminal 7 kDa peptide. The mature 22 kDa TIP3;1 protein accumulated in the tonoplast when the synthesis of precursor had ceased and its mRNA had disappeared. TIP3;1 aquaporins prevail in tonoplasts and form efficient water channels. Along with TIP3;1, other TIPs, namely TIP2 and TIP1;1, were found in pea seeds (Jauh et al., Reference Jauh, Philips and Rogers1999), and TIP2 was reported in mature horse chestnut seeds (Obroucheva et al., Reference Obroucheva, Lityagina, Novikova and Sinkevich2012). The transcripts of TIP3;1, TIP3;2 and TIP5;1 genes have been identified in dry seeds of Arabidopsis (Willigen et al., Reference Willigen, Postaire, Tournaire-Roux, Boursiac and Maurel2006), whereas in Vicia faba minor seeds the embryo axes contained TIP3;1, TIP3;2, TIP1;1, TIP2;1 and TIP2;2 transcripts (Novikova et al., Reference Novikova, Tournaire-Roux, Sinkevich, Lityagina, Maurel and Obroucheva2013).

Such TIP-formed water channels could provide water inflow to the enlarging vacuoles in maturing seeds and, later on, during vacuole transformation to protein-storage vacuoles, and they could participate in water outflow, resulting from water displacement by deposited reserve proteins. This possibility follows from the property of water channels allowing water to pass in both directions.

Information about PIPs during seed maturation is still lacking. Nevertheless, dry seeds are known to contain a number of aquaporins, apparently preformed in developing and maturing seeds. In dry Arabidopsis seeds, 11 PIP isoforms were found (Willigen et al., Reference Willigen, Postaire, Tournaire-Roux, Boursiac and Maurel2006), as in dry rice seeds (Liu et al., 2007). In the latter, the transcripts of some PIP genes were also present, mainly PIP2;7 mRNAs, whereas mRNAs of PIP1;1, PIP1;2 and PIP2;1 were not abundant. At the same time, dry Arabidopsis seeds contained mRNAs of TIP3;1, TIP3;2 and TIP5;1 genes (Willigen et al., Reference Willigen, Postaire, Tournaire-Roux, Boursiac and Maurel2006). TIP1;1 and TIP2;1 were absent from the embryos of Arabidopsis dry seeds, but were present in the seed coat (Gattolin et al., Reference Gattolin, Sorieul and Frigerio2011).

In the embryonic axes, where germination events will later develop, the presence of constitutive aquaporin genes was confirmed. In the axes of dry broad bean seeds, PIP2;1, NIP1;1, TIP3;1; TIP3;2, TIP1;1, TIP2;1 and TIP2;2 genes were identified (Novikova et al., Reference Novikova, Tournaire-Roux, Sinkevich, Lityagina, Maurel and Obroucheva2013). Preformed aquaporins PIP1, PIP2, TIP3;1 and TIP2 were detected in embryonic axes of horse chestnut seeds at shedding, during stratification and dormancy release (Obroucheva et al., Reference Obroucheva, Lityagina, Novikova and Sinkevich2012). Therefore, the embryonic axes are equipped with the aquaporin machinery at the commencement of germination. Seed germination occurs by elongation of embryonic axis cells, triggered by water inflow during imbibition (Obroucheva, Reference Obroucheva2012). Aquaporin gene expression is low during seed imbibition and only starts when a hydration level of 50–55% (fresh weight) is achieved (Obroucheva, Reference Obroucheva2012). The main changes in aquaporin composition are initiated at radicle emergence and just after it, when the expression of PIP and TIP genes is strongly activated. Arabidopsis seeds have shown enhanced expression of PIP1;1, PIP1;2, PIP1;4, PIP2;1, PIP2;2, PIP2;6 and PIP2;7 genes (Willigen et al., Reference Willigen, Postaire, Tournaire-Roux, Boursiac and Maurel2006). In germinating rice seeds, PIP1;1 and PIP1;3 genes were mostly transcribed (H.Y. Liu et al., Reference Liu, Yu, Cui, Sun, Sun, Tang, Kwak and Su2007; C. Liu et al., Reference Liu, Fukumoto, Matsumoto, Gena, Frascaria, Kaneko, Katsuhara, Zhong, Sun, Zhu, Iwasaki, Ding, Calamita and Kitagawa2013) whereas in the embryonic axes of broad bean seeds, only PIP2;1 expression was observed. Along with NIP1;1, these genes demonstrated noticeably stable expression of their transcripts; PIP1 transcripts were not identified (Novikova et al., Reference Novikova, Tournaire-Roux, Sinkevich, Lityagina, Maurel and Obroucheva2013). Apparently, these aquaporins form water channels, which, if opened, can provide sufficient water entry into embryo axis cells that are beginning to elongate.

Cell elongation is closely related to vacuole formation and enlargement because the vacuoles represent an osmotic compartment responsible for osmotically driven water inflow. Cell vacuolation precedes the beginning of elongation (Obroucheva, Reference Obroucheva2012; Novikova et al., Reference Novikova, Tournaire-Roux, Sinkevich, Lityagina, Maurel and Obroucheva2013). In orthodox seeds, vacuolation occurs in the course of restoration of protein-storage vacuoles in embryo cells. Due to the step-by-step proteolysis of reserve proteins deposited in protein-storage vacuoles, these are transformed back to vacuoles. In broad bean seeds, which are characterized by hypogeal germination, such vacuole biogenesis occurs mainly in the hypocotyl, which is the first to elongate (Novikova et al., Reference Novikova, Tournaire-Roux, Sinkevich, Lityagina, Maurel and Obroucheva2013). In root cells produced de novo by the root meristem after radicle emergence, vacuoles are formed from provacuoles that are developed in the lagoons of the endoplasmic reticulum. Both patterns of vacuole biogenesis in embryonic axes lead to vacuole fusion and enlargement prior to the beginning of cell elongation, and culminating in the formation of a central vacuole after radicle emergence, i.e. in rapidly elongating cells. Such vacuolar dynamics is closely related to TIP composition. First of all, it has been observed that the expression of TIP3;1 and TIP3;2 genes ceased after radicle emergence, and these proteins disappeared in parallel with the transformation of protein-storage vacuoles to vacuoles (Novikova et al., Reference Novikova, Tournaire-Roux, Sinkevich, Lityagina, Maurel and Obroucheva2013). Just after radicle emergence, the greatly increased expression of TIP1;1, TIP2;1, TIP2;2 isoforms is closely related to accelerated water inflow to rapidly elongating cells.

This trend, which was analysed in embryo axes of broad bean seeds, is supported by observations in other seed species. The cessation of TIP3 gene expression and progressive disappearance of these proteins after germination was demonstrated in Arabidopsis seeds (Willigen et al., Reference Willigen, Postaire, Tournaire-Roux, Boursiac and Maurel2006; Hunter et al., Reference Hunter, Craddock, Benedetto, Roberts and Frigerio2007; Gattolin et al., Reference Gattolin, Sorieul and Frigerio2011) and rice aleurone cells (Takahashi et al., Reference Takahashi, Rai, Kitagawa, Morita and Masumura2004; Li et al., Reference Li, Peng, Yu, Zhang, Cai, Sun and Su2008). As protein-storage vacuoles were absent from horse chestnut embryonic axes, no TIP3;1 degradation occurred and TIP3;1 remained in the tonoplast at radicle emergence, along with TIP2 (Obroucheva et al., Reference Obroucheva, Lityagina, Novikova and Sinkevich2012).

The active expression of TIP1 subtype was correlated with cell growth (Ludevid et al., Reference Ludevid, Höfte, Himelblau and Chrispeels1992; Hunter et al., Reference Hunter, Craddock, Benedetto, Roberts and Frigerio2007; Gattolin et al., Reference Gattolin, Sorieul, Hunter, Khonsari and Frigerio2009). It started 2 d after radicle emergence (Takahashi et al., Reference Takahashi, Rai, Kitagawa, Morita and Masumura2004; Willigen et al., Reference Willigen, Postaire, Tournaire-Roux, Boursiac and Maurel2006; Hunter et al., Reference Hunter, Craddock, Benedetto, Roberts and Frigerio2007; Li et al., Reference Li, Peng, Yu, Zhang, Cai, Sun and Su2008; Gattolin et al., Reference Gattolin, Sorieul and Frigerio2011), although TIP1;1 was previously absent from Arabidopsis embryos (Willigen et al., Reference Willigen, Postaire, Tournaire-Roux, Boursiac and Maurel2006; Hunter et al., Reference Hunter, Craddock, Benedetto, Roberts and Frigerio2007). The enhanced expression of TIP1;1 at the transcript level and accumulation of TIP proteins in rapidly elongating cells (Novikova et al., Reference Novikova, Tournaire-Roux, Sinkevich, Lityagina, Maurel and Obroucheva2013) have confirmed the growth-related changes in TIP composition.

TIP2 isoforms are the next to be actively expressed in growing organs. Their expression is enhanced a bit later than the expression of TIPs. For example, in Arabidopsis embryos TIP2 gene expression increased from 2 d after radicle emergence (Willigen et al., Reference Willigen, Postaire, Tournaire-Roux, Boursiac and Maurel2006), after the enhancement of TIP1;1 expression, although TIP2 proteins seemed to be virtually absent from the embryos (Gattolin et al., Reference Gattolin, Sorieul and Frigerio2011). In rice seedlings, TIP2;2 expression increased on the fifth day after germination (Li et al., Reference Li, Peng, Yu, Zhang, Cai, Sun and Su2008). According to Hunter et al. (Reference Hunter, Craddock, Benedetto, Roberts and Frigerio2007), expression of TIP2s is typical of mature conductive tissues, i.e. of fully elongated cells. Turning back to our data (Novikova et al., Reference Novikova, Tournaire-Roux, Sinkevich, Lityagina, Maurel and Obroucheva2013), a sharp rise in TIP2;1 and TIP2;2 gene expression recorded in 2-cm long embryonic axes coincided with the promoted cell elongation in broad bean roots, and almost completed cell elongation in hypocotyls, which indicated a close relationship between TIP2 accumulation, accelerated water uptake, enlargement of central vacuoles and the transition of fully elongated cells to maturation.

Therefore, the changes in tonoplast aquaporin composition in embryonic axes are closely related to the patterns of vacuole biogenesis before the initiation of cell elongation. Subsequently, the contribution of vacuoles to cell elongation is due to the synthesis of TIP1 and TIP2 aquaporins and apparently to a greater presence of water channels in the tonoplasts. In all growing plant organs, cell proliferation is associated with plasmalemma aquaporins, whereas cell elongation is related to tonoplast aquaporins as well (Obroucheva and Sinkevich, Reference Obroucheva and Sinkevich2010).

An adequate equipment of growing embryonic axis cells with aquaporins is a prerequisite, but still not an assurance, of water-channel participation in water transport, because the channels can be either open (active) or closed (inactive). For studying water-channel activity, the method developed for roots is suitable (Barrowclough et al., Reference Barrouclough, Peterson and Steudle2000; Javot and Maurel, Reference Javot and Maurel2002). It consists of water-uptake measurements and treatments with mercurials, which block water channels, with subsequent recovery from mercury inhibition by reducing agents. This method evaluates the water permeability of both cell membranes, but primarily of the plasmalemma. Such experiments with horse chestnut (Obroucheva et al., Reference Obroucheva, Lityagina, Novikova and Sinkevich2012) and broad bean (Novikova et al., Reference Novikova, Tournaire-Roux, Sinkevich, Lityagina, Maurel and Obroucheva2013) embryonic axes, both growing by cell elongation, have shown that at radicle emergence water channels are closed, whereas in growing axes they are open. In imbibing Arabidopsis seeds also, no inhibiting effect of mercury ions was observed until the onset of growth (Willigen et al., Reference Willigen, Postaire, Tournaire-Roux, Boursiac and Maurel2006), apparently due to the closed state of water channels. Pea seeds, imbibed in the presence of mercury ions, did not display a change in the rate of water absorption (Veselova and Veselovsky, Reference Veselova and Veselovsky2006), which indicates a closed state of water channels. This data can be interpreted in the following way: water uptake until radicle emergence occurs only by diffusion, without any participation of water channels, whereas aquaporin-mediated water inflow in embryo axes begins only when cell elongation is enhanced. According to Veselova and Veselovsky (Reference Veselova and Veselovsky2006), such slow diffusion of water during imbibition prevents seed hydration from occurring too rapidly, which could result in hypoxia. In ageing pea seeds, the water channels were open, which was evident from accelerated seed imbibition in the presence of mercurials; this effect was explained by damage to the dephosphorylation system and thus the inability to close the water channels (Veselova and Veselovsky, Reference Veselova and Veselovsky2006).

The functioning of open water channels in growing embryo axes is closely related to the greatly enhanced water entry into rapidly elongating cells. Their operation occurs in parallel with continuing slow diffusion of water. Accelerated water inflow through water channels is driven by the accumulation of endogenous osmotic solutes in elongating cells due to sucrose import from storage tissues or organs. Therefore, although dry seeds contain the transcripts of aquaporin genes and aquaporins themselves, the operation of water channels starts only after radicle protrusion, to provide enhanced water inflow to growing seedling cells.