Results and discussion

Vicia faba beans belong to the class of orthodox seeds, that is, can be stored dry and rapidly absorb available water to complete germination. Figure 1 shows the time course of the main physiological events in imbibing seeds as related to WC of the embryonic axes. The WC curve displays the three typical phases: slow water uptake (Phase I for 19 h, WC range of 10–62%), more rapid water inflow (Phase II, 19–25 h, WC range of 62–70%) and Phase III characterized by active water uptake due to the cell elongation initiated in embryonic axes after 25 h of imbibition. Cell elongation starts in embryonic axes much earlier than cell division, which only begins after radicle protrusion.

Fig. 1. Activation of PM H⁺-ATPase in embryo axes of imbibing Vicia faba seeds. Dependence of enzyme activity in embryonic axes on their water content, as related to growth initiation. Activation of the enzyme occurred prior to growth commencement, at a water content between 50 and 60%, on average at 55% (arrow). Three concentrations of PEG were applied to control this value. Points of water uptake after incubation in PEGs are shown by dashed lines, and activation of enzyme or its absence is indicated. Cell elongation was evaluated by the rate of radicle emergence, which started at 25 h after imbibition (long arrow), and ended at 43–44 h, when all viable seeds showed radicle protrusion. Initiation of cell division (small arrows) was recorded by the appearance of mitotic figures after DNA staining of embryo axis sections with Schiff reagent.

The WC dynamics was compared with the capacity of embryonic axes to acidify the ambient solution, that is, with the activity of PM H⁺-ATPase (Fig. 2A). PM H⁺-ATPase activity sharply appeared in the embryo axes imbibed for 18 h. We tested also the response of PM H⁺-ATPase in embryonic axes to fusicoccin (Fig. 2B), a specific activator and stabilizer of this enzyme (Camoni et al., Reference Camoni, Visconti and Aducci2013). Fusicoccin exerted a pronounced stimulating effect on H⁺ efflux. Thus, embryonic axes contain typical PM H⁺-ATPase. This enzyme belongs to the P-type ATPases as its activity is inhibited by o-vanadate, imitating phosphate analogue in the ATP hydrolysis cycle (Fig. 3).

Fig. 2. (A) Activity of PM H⁺-ATPase in embryonic axes of imbibing Vicia faba seeds. (B) Effect of 10⁻⁶ M fusicoccin on PM H⁺-ATPase activity.

Fig. 3. Identification of PM H⁺-ATPase and 14-3-3 proteins in embryonic axes of Vicia faba seeds. 1: before 18 h imbibition; 2: after 18 h imbibition; 3: at radicle emergence; 4: after radicle emergence.

For convenience, we indicated on the WC curve the threshold of enzyme activation by an arrow (Fig. 1) at 18 h imbibition and WC of 55%. To be sure that activation occurred at this WC, a series of treatments with the osmoticum PEG-6000 at various concentrations was performed. 30% PEG permitted the axes to imbibe to 39 ± 1%, in 25% PEG embryonic axes had a WC of 50 ± 1.5%, and at 20% PEG, WC was held at of 60 ± 2%. After incubation in 30% PEG, WC in embryonic axes was only 38%, and no PM H⁺-ATPase activity was recorded (Fig. 1). The same situation was observed with 25% PEG, which confined 50% WC in embryonic axes without any effect on H⁺ extrusion. Only at 20% PEG maintaining 60% WC in embryonic axes, the activation of PM H⁺-ATPase was demonstrated. These data confirm that PM H⁺-ATPase is activated by inflowing water when WC in imbibing axes reached 55%. The activation events occurred prior to cell elongation commencement.

PM H⁺-ATPase has been identified in developing Arabidopsis and wheat seeds (Harper et al., Reference Harper, Manney and Sussman1994; Rober-Kleber et al., Reference Rober-Kleber, Albrechtova, Fleig, Huck, Michalke, Wagner, Speth, Neuhaus and Fischer-Iglesias2003). Apparently, the enzyme is stored during seed maturation because, in our experiments, embryonic axes incubated in an α-amanitin solution (1 μg ml⁻¹) are capable to germinate and extrude H⁺ ions; this means that H⁺ ion efflux is related to stored mRNAs of PM H⁺-ATPase. In maize embryos (Sanchez-Nieto et al., Reference Sanchez-Nieto, Enriquez-Arredondo, Guzman-Chavez, Hernandez-Murioz, Ramirez and Gavilanes-Ruiz2011), it was shown that after 5 h of imbibition, the enzyme differs from that in dry seeds by a number of biochemical properties, including the sensitivity to o-vanadate, trypsin and denaturing temperatures. These observations are in agreement with the conclusion about hydration-driven activation of the enzyme, but 5 h of imbibition provides a too low hydration for activation of PM H⁺-ATPase.

In imbibing embryonic axes from bean seeds, the presence of PM H⁺-ATPase protein was confirmed immunochemically with a specific antibody. Figure 4 shows the bands of this enzyme during imbibition. Concurrently, the presence of 14-3-3 protein was demonstrated. These data showed the presence of the enzyme in embryonic axes capable to be activated at 55% FW during imbibition.

Fig. 4. Effect of o-vanadate on H⁺ ion efflux from embryonic axes of Vicia faba seeds.

The following explanation of the role of high water content in PM H⁺-ATPase activation can be put forward. This high-molecular weight intrinsic protein has a tertiary structure characterized by the interaction of functional domains and conformational changes within them leading to protein functioning. For successful H⁺ transport from the cytoplasm to the apoplast, the movement across the hydrophobic plasma membrane is a critical event (Buch-Pedersen et al., Reference Buch-Pedersen, Pedersen, Veierskov, Nissen and Palmgren2009). H⁺ enters the PM as a hydrated molecule and moves along a ‘protein wire’, a chain of charged water molecules, jumping from one molecule to another towards Asp684, its acceptor/donor (Buch-Pedersen and Palmgren, Reference Buch-Pedersen and Palmgren2003; Guerra and Bondar, Reference Guerra and Bondar2015). Asp684 is located near the central water cavity of 80 A³ volume filled with 10–16 water molecules; it represents a unique structure specific for H⁺-ATPase absent in other ATPases of the P-type (Buch-Pedersen et al., Reference Buch-Pedersen, Pedersen, Veierskov, Nissen and Palmgren2009). Due to conformational changes of Asp486 and Arg655 positioned at the opposite sites of water cavity, H⁺ enters the water cavity, which then changes its configuration and opens at the extracellular site of the PM. Such H⁺ pathway from hydrophilic cytoplasmic domains through a hydrophobic transmembrane domain needs high water content and conformational transitions coupled to ATP hydrolysis.

To check the role of phosphorylation, we measured its rate by Pi accumulation (Fig. 5). It increased at enzyme activation at 18 h and is even more active later, when the activity of PM H⁺-ATPase rises. Therefore, the enzyme hydration-driven activation is accompanied by activation of ATP hydrolysis. This conclusion was confirmed by the action of o-vanadate on the capacity of embryonic axes to extrude H⁺ ions (Fig. 3) because this inhibitor interferes with the hydrolysis of ATP by substitution for phosphate.

Fig. 5. H⁺-ATPase activity measured by ATP hydrolysis in imbibing embryonic axes of Vicia faba. The experiments were done in triplicates. Activation time 18 h, time of radicle protrusion 36 h. Arrow indicates germination.

Hydration-activated enzyme acquires its proper confirmation and is not in short of substrate delivery. After enzyme activation, H⁺ efflux continues, supported by phosphorylation of penultimate threonine or some other amino acid residues located in the regulatory domain of the enzyme molecule (Falhof et al., Reference Falhof, Petersen, Fuglsang and Palmgren2016). 14-3-3 protein is an activator of phosphorylation and fusicoccin is its stabilizer (Camoni et al., Reference Camoni, Visconti and Aducci2013). Along with this regulatory mechanism, a phosphorylation-independent pathway can operate via 14-3-3 binding to the phosphoprotein (Borch et al., Reference Borch, Bych, Roepstorff, Palmgren and Fuglsang2002; Fuglsang et al., Reference Fuglsang, Borch, Bych, Jahn and Roepstorff2003). In the case of Vicia faba, the regulation of enzyme activity after its hydration-driven activation is performed in the regulatory domain via its interaction with 14-3-3 protein and endogenous fusicoccin. We have shown that 14-3-3 proteins (Fig. 4) are concurrently present with the enzyme in imbibing embryonic axes of Vicia faba; furthermore, the endogenous fusicoсcin ligands were previously discovered in Vicia faba minor embryonic axes (Antipova et al., Reference Antipova, Bartova, Kalashnikova, Obroucheva, Voblikova and Muromtsev2003).

Rapid activation of PM H⁺-ATPase is one of a series of water-triggered metabolic systems in imbibing embryonic axes. Previously, threshold WC levels were demonstrated for activation of glycolysis, TCA cycle and amino acid metabolism (20–23%), for activation of respiration rate due to completion of mitochondriogenesis (45%), for proteolysis in protein-storing vacuoles (51–55%) and amylolysis in amyloplasts (45%), for initiation of ribosomal protein synthesis (52–55%) and gene transcription (42–47%) (Obroucheva, Reference Obroucheva2012). As a result, successive activation of main metabolic systems abundant in dry seeds occurs in embryonic axes of orthodox seeds due to the imbibition up to threshold WC levels. The importance of such activation of protein synthesis has been confirmed by Galland et al. (Reference Galland, Huguot, Arc, Cueff, Job and Rajjou2014) with imbibing Arabidopsis seeds incubated in labelled methionine solutions. They identified a bulk of non-labelled (not de novo synthesized) proteins mainly during the first 8 h of imbibition. Apparently, these proteins were synthesized from amino acids produced by amino acid metabolism activated at low WC.

It is worth noting some specific features of such hydration-driven activation. In orthodox seeds, it occurs automatically, because a level of 60% WC is achieved even in dead seeds, which absorb water as any capillary-porous body containing a high number of hydrophilic sites (Obroucheva et al., Reference Obroucheva, Sinkevich, Lityagina and Novikova2013). In viable seeds, de novo gene transcription can start actually at WC exceeding 60% and is supported by activated translation. Hydration-driven activation of existing main metabolic systems in combination with gene transcription at high WC can be considered as a tool providing rapid seed germination and its correlation with available water.

Investigation of enzyme activation raises the question of discrimination between the terms ‘enzyme activation’ and ‘enzyme activity’. After hydration-driven activation, PM H⁺-ATPase continues to be active up to radicle emergence and including it, remains high after radicle emergence, during further cell elongation growth. Its function is the maintenance of acid growth in seedling organs. The importance of PM H⁺-ATPase is not limited to growth initiation at radicle emergence, that is, to germination, but it continues to maintain acid growth in the growing seedling. It can be exemplified by hypocotyl elongation in Arabidopsis (Takahashi et al., Reference Takahashi, Hayashi and Kinoshita2012; Hayashi et al., Reference Hayashi, Takahashi, Inoue and Kinoshita2014), by coleoptile growth in maize seedlings (Rudashevskaya et al., Reference Rudashevskaya, Kirpichnikova and Shishova2005) and by roots of Arabidopsis (Planes et al., Reference Planes, Ninoles, Rubio, Rissoli, Rueso, Garcia-Sanchez, Alejandro, Gonzales-Guzman, Hedrich, Rodriguez, Fernandez and Serrano2015).

As the activity of PM H⁺-ATPase can be stimulated by auxin (Takahashi et al., Reference Takahashi, Hayashi and Kinoshita2012; Arsuffi and Braybrook, Reference Arsuffi and Braybrook2018) and inhibited by abscisic acid (Planes et al., Reference Planes, Ninoles, Rubio, Rissoli, Rueso, Garcia-Sanchez, Alejandro, Gonzales-Guzman, Hedrich, Rodriguez, Fernandez and Serrano2015), the next question was to clarify whether the target of ABA and auxin is the activation of the enzyme or the activity of the enzyme after its activation. For this purpose, in imbibing Vicia faba embryonic axes (Fig. 6A,C) the effects of IAA and ABA were estimated at a WC of 52–54%, that is, slightly below the WC threshold for enzyme activation, and at a WC of 63–65%, when the enzyme is already active. Auxin did not significantly stimulate either the activation or activity of PM H⁺-ATPase within the concentration range of 10⁻⁵–10⁻⁷ M, which corresponds to the auxin-induced doubling of acid growth in elongating hypocotyls (Takahashi et al., Reference Takahashi, Hayashi and Kinoshita2012). In our experiments, IAA did not promote the elongation of embryonic axes as followed from their fresh weight data. Germination of Vicia faba seeds and activity of PM H⁺-ATPase just after germination appear to be auxin-independent which may be due to the inability of embryonic axes to accept exogenous auxin because mature Vicia faba seeds lack endogenous auxins and do not synthesize them after radicle emergence (Plesse et al., Reference Pless, Böttger, Hedden and Graebe1984; Sztein et al., Reference Sztein, Ilic, Cohen and Cooke2002).

Fig. 6. Effect of ABA and IAA on activation and activity of PM H⁺-ATPase in embryo axes of imbibing Vicia faba seeds. (A,C) Before activation of PM H⁺-ATPase and (B,D) after activation of PM H⁺-ATPase. (A,B) Effect of ABA and (C,D) effect of IAA.

Similar experiments were carried out with ABA (Duchefa Biochemie, The Netherlands) at concentrations of 10⁻⁵–10⁻⁷ M (Fig. 6). At a WC below the threshold level, enzyme activation and growth were inhibited at 10⁻⁵ M ABA, but no inhibition was recorded at lower concentrations (Fig. 6A). At early imbibition, we must take into account the active accumulation of transcripts of 8′-hydroxylase ABA, the enzyme responsible for ABA degradation (Kimura and Numbara et al., Reference Kimura and Nambara2010). As a result, the level of endogenous ABA drops to 10⁻⁸–10⁻⁹ M and remains low. In the presence of toxic 10⁻⁵ M exogenous ABA, synthesized 8′-hydroxylase ABA apparently cannot minimize the inhibitory action of ABA on growth and activation of PM H⁺-ATPase. At lower 10⁻⁶–10⁻⁷ M ABA, 8′-hydroxylase ABA could destroy it, counteracting the ABA inhibitory effect on H⁺ efflux. Such an approach permits to conclude that 10⁻⁶ and 10⁻⁷ M ABA did not inhibit the activation of PM H⁺-ATPase.

Already activated enzyme (Fig. 6B) was partially inhibited at all ABA concentrations. Such inhibition occurs against a background of residual ABA 8′-hydrolase synthesis, which could not counteract the inhibitory action of ABA on the activity of H⁺-ATPase and germination. These data correspond to ABA-induced growth inhibition (Hayashi et al., Reference Hayashi, Takahashi, Inoue and Kinoshita2014; Planes et al., Reference Planes, Ninoles, Rubio, Rissoli, Rueso, Garcia-Sanchez, Alejandro, Gonzales-Guzman, Hedrich, Rodriguez, Fernandez and Serrano2015) observed in seedling organs at high WC.

To conclude, auxin did not stimulate activation and activity after activation of PM H⁺-ATPase, whereas ABA inhibited only the enzyme activity after its activation, which seems to be the target of hormonal regulation of this enzyme.

It is quite possible that the regulation of PM H⁺-ATPase in dormant seeds is more complicated. In horse chestnut seeds, which are characterized by a long deep physiological dormancy during winter, the enzyme activity was recorded during the whole dormant period and dormancy release. These seeds, like other recalcitrant seeds, have high WC, embryonic axes in dormant seeds have a WC of 62%, and germination needs a WC of 72% (Obroucheva et al., Reference Obroucheva, Sinkevich and Lityagina2016). Apparently, the enzyme is already activated. However, the activity changed along the course of dormancy: the first 5 weeks, that is, during embryo dormancy, the activity is weak and not sensitive to fusicoccin, but during the following 10 weeks (coat-imposed dormancy) it increased and was stimulated by fusicoccin (Obroucheva et al., Reference Obroucheva, Lityagina and Sinkevich2018). It is tempting to speculate that in dormant seeds the enzyme is at first in an autoinhibited state (Portillo, Reference Portillo2000). In sunflower embryos at low WC (10%), the measurement of membrane potential in the radicle epidermis of dormant and non-dormant seeds has shown that in dormant seeds, the enzyme was not active although it was stimulated by fusicoccin (De Bont et al., Reference De Bont, Naim, Arbelet-Bonnin, Xia, Palm, Meimoun, Mancuso, El-Maarouf-Bouteau and Bouteau2019). The authors assumed that in dormant seeds, the enzyme was inhibited by ABA and that dormancy release is related to ethylene and ROS compounds. Therefore, the state of the enzyme and its regulation in dormant seeds needs further investigation.