H+-ATPases Regulated by Auxin and ABA Mediate Acid Growth of Soybean Embryonic Axis During Germination

Abstract

Soybean seeds (Glycine max) are of great economic and nutritional importance due to their high oil and protein content. Seed germination is an essential process that influences crop yield and quality. The seed embryo resumes growth when it imbibes, which induces the expression of genes related to cell expansion. The role of acid growth in the embryonic axis during germination is not well characterized. Thus, the aim of this study was to verify the contribution of acid growth in the soybean embryonic axis germination. Acid growth is mediated by the acidification of extracellular medium due to the action of H+-ATPases, which activate expansins. We found that the expression of expansins was significantly increased throughout the germination. The expression of H+-ATPases was significantly increased at 3 and 24 h after imbibition (HAI), with major pumping activity at 24 HAI. The auxin and ABA signaling cascades during soybean germination suggest that these hormones are involved with the regulation of H+-ATPase in germinating soybean. To verify the influence of auxin and ABA on H+-ATPase functioning during germination, we treated seeds with IAA, 2,4D, ABA, and ATPase inhibitor, and germinated them in purple agar medium. We observed that IAA, 2,4D, and ABA affected H+-ATPase functioning, by delaying or inhibiting soybean germination. Our results indicate the role of acid growth controlled by H+-ATPase and its regulators—auxin and ABA—in the soybean embryonic axis during germination.

1. Introduction

Legumes are of great importance in human and animal nutrition. Glycine max (L.) Merrill, commonly known as soybean, is one of the most important crops in the world for its high oil and protein content, with about 40% oil, 20% protein, 35% carbohydrates, and 5% minerals [1]. Seed germination is an important process that influences crop yield and quality. Germination begins with the imbibition of water by the seed, at a level sufficient to trigger the metabolism and growth of the embryo, facilitating cell expansion and protrusion of the embryonic axis [2].

The germination process is influenced by environmental factors, such as the availability of adequate levels of water, light, humidity, temperature, and oxygen, as well as by the action of endogenous phytohormones. In the past decades, the antagonistic action of abscisic acid (ABA) and gibberellins (GAs) has been related to many plants’ developmental processes, including those occurring in seeds. The ABA/GA balance can release seeds from dormancy, promoting germination [3]. Acting synergistically with ABA, auxin controls seed dormancy by recruiting AUXIN RESPONSE FACTOR 10 and 16 to induce the transcription of ABSCISIC ACID INSENSITIVE3 (ABI3), a negative regulator of seed germination [4]. For instance, seeds with over-production of auxin (iaaM-OX) showed delayed germination, whereas seeds with low amounts of auxin (yuc1/yuc6) increased germination [5]. The downregulation of ARF10 by miR160 is also critical for Arabidopsis germination [6]. Moreover, Ref. [7] showed that an auxin signaling repressor protein (IAA8) was capable of promoting seed germination by downregulating ABI3.

During soybean seed germination, the embryonic axis shows dry biomass decrease and major cellular expansion [8]. To drive this expansion, the pathways responsible for the extension of the cell wall are activated in the embryonic axis. Key cell wall genes were highly expressed at specific germination stages and correlate with structural cell wall changes required for posterior protrusion of the embryonic axis [8,9]. Two expansin genes were found to be most expressed in soybean seeds at the embryonic axis and at 12 h of water incubation [8]. Montechiarini et al., 2020 [9] showed that one of these expansin genes was induced in the elongation zone of the embryonic axes during soybean germination in water and repressed by ABA treatment.

Recent studies have highlighted the loosening of the cell wall mediated by acid growth, through the auxin/H+-ATPase/expansin signaling cascade [10,11,12]. The plasma membrane H+-ATPase (PM H+-ATPase) functioning can be activated by the action of auxin in certain developmental stages. In the auxin signaling pathway, the expression of SMALL AUXIN UP-RNA (SAUR) genes is induced by the regulation of AUX1->TIR1/AFB->AUX-IAA->ARF, which in turn will inhibit the action of phosphatase type 2C subfamily D (PP2C-D) [10,11]. The PM H+-ATPase will remain phosphorylated on penultimate threonine residue (Thr-947), allowing the binding of the 14-3-3 protein and activation of the proton pumping [10]. In Arabidopsis thaliana, ABA inhibit the activity of PM H+-ATPase through SNF1-RELATED PROTEIN KINASE—SnRK2.2 or SnRK2.3—phosphorylation of unidentified residue on its C-terminal, which affects seed germination [13]. Depending on the timing of their increases in the embryonic axis, auxin and ABA can modulate PM H+-ATPase activity in distinct ways [14].

In this study, we investigated the role of plasma membrane H+-ATPase and its hormonal regulators, auxin and abscisic acid (ABA), in the expansion of the embryonic axis during the sensu stricto germination of soybean seeds. By combining gene expression analyses and physiological assays, we aimed to understand how acid growth mechanisms contribute to early seedling development. Our findings shed light on the H+-ATPase activity and its relevance to the germination process in soybean.

2. Materials and Methods

2.1. Plant Material

Soybean seeds of cultivar BRS 284 were disinfected for 1 min in 2% hypochlorite and then washed with sterile distilled water. The seeds were germinated in Petri dishes (9 cm × 9 cm), containing 12 mL of distilled water and 3 sheets of filter papers. The plates were placed in a growing chamber with temperature at 28 °C under a photoperiod of 12/12 h for 24 h.

2.2. Expression Analysis of Soybean Genes Involved with Acid Growth

2.2.1. Transcriptome Database Analysis

The expression data of the expansin and PM H+-ATPase genes in the soybean embryonic axis on dry seeds and at 3, 6, 12, and 24 h after imbibition (HAI) were retrieved from Soybean Expression Atlas v2 [15]. The expansin expression data were plotted using R package gplots 3.2.0 heatmap 2 function with transcripts per million (TPM) values. For the auxin- and ABA-related genes, the signaling cascades of these two hormones—ko04075—were downloaded from the KEGG pathway. Based on the Arabidopsis orthologs, we filtered the soybean genes with differential expression on the embryonic axis during germination [16]. The expression values were log2 transformed and plotted using the online tool Heatmapper (http://www.heatmapper.ca/expression/) (accessed on 2 December 2024).

2.2.2. RNA Extraction, cDNA Construction, and RT-qPCR

Soybean seeds were germinated as described in plant material. Total RNA was isolated from embryonic axes at 3, 6, 12, and 24 HAI using Trizol (Invitrogen, Waltham, MA, USA) and according to the manufacturer’s instructions. RNA quality was measured using a a NanoDrop™ One spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RNA was also verified by 1% agarose gel electrophoresis with ethidium bromide staining. The synthesis of cDNA was performed with 5 μg of RNA using the GOscript kit (Promega, Madison, WI, USA). The GmEXP37 gene expression was evaluated by RT-qPCR with SYBR Green PCR Master Mix (Applied Biosystems, Waltham, MA, USA), using the QuantStudio Real-Time PCR System (Applied Biosystems, Waltham, MA, USA)) and the following primers: GmEXPA37_F-GCCCTTCCTAACAACAATGG and GmEXPA37_R-CATGGAACCCTTTGAAATAGC. Standard thermal cycling conditions included initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing at 60 °C for 1 min. The relative expression of GmEXPA37 was quantified using 2(−ΔCt).

2.3. Plasma Membrane H+-ATPase Activity Analysis

2.3.1. Preparation of Membrane Vesicles

Soybean seeds were germinated as described in plant material. The embryonic axes were collected at 3, 6, 12, and 24 h after imbibition (HAI). Membrane vesicles were prepared from soybean embryonic axes using a differential centrifugation protocol based on the method described by [17], with slight modifications. Approximately 200 embryonic axes were ground in a mortar and pestle using an ice-cold extraction buffer (2 mL per gram of fresh tissue) composed of 250 mM sucrose, 10% (v/v) glycerol, 0.5% (v/v) polyvinylpyrrolidone (PVP-40), 5 mM EDTA, 0.3% (w/v) BSA, and 100 mM Tris-HCl (pH 7.6). Before use, the buffer was supplemented with 150 mM KCl, 3.3 mM DTT, 1 mM PMSF, and 1 mM benzamidine. The homogenate was filtered through four layers of cheesecloth and subjected to sequential centrifugation steps: first at 1500× g for 15 min, then at 10,000× g for 20 min, and finally at 100,000× g for 45 min. The resulting pellet was gently resuspended in a cold buffer containing 10 mM Tris-HCl (pH 7.6), 10% (v/v) glycerol, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, and 1 mM benzamidine. All steps were carried out at 4 °C. The vesicle preparations were snap-frozen in liquid nitrogen and stored at −80 °C until use. Protein concentrations were determined following the method described by [18].

2.3.2. Enzyme Assay

The hydrolytic activity of P-type H+-ATPase was assessed through a colorimetric method that quantifies the release of inorganic phosphate (Pi), as described by [19]. Reactions were carried out in a buffer containing 50 mM Tris-HCl (pH 6.5), 3 mM MgSO₄, 100 mM KCl, 0.2 mM NaMoO₄, and 1 mM ATP. The enzymatic reaction was initiated by adding membrane protein (final concentration of 100 µg mL⁻¹) and incubated for 30 min at 25 °C. To terminate the reaction, 5% (w/v) trichloroacetic acid, pre-chilled on ice, was added. Hydrolytic activity specific to plasma membrane H+-ATPase was determined by measuring vanadate-sensitive Pi release using 0.2 mM Na₃VO₄ as a selective inhibitor.

2.3.3. Proton Pumping Assay

ATP-dependent proton translocation in membrane vesicles was evaluated by monitoring the quenching of the fluorescent dye 9-amino-6-chloro-2-methoxyacridine (ACMA) at 25 °C using a Hitachi F-3010 fluorometer, following a modified version of an established protocol. Fluorescence was recorded at 485 nm (emission) with excitation at 415 nm. The assay buffer consisted of 10 mM Tris-HCl (pH 6.5), 2 µM ACMA, 5 mM MgSO₄, 100 mM KCl, and 1 mM ATP. Proton transport was initiated by adding membrane vesicles (100 µg mL⁻¹ final concentration). The collapse of the generated proton gradient and restoration of fluorescence were induced by the addition of 20 mM NH₄Cl. As with the hydrolytic assay, 0.2 mM Na₃VO₄ was used to distinguish the PM H+-ATPase-specific activity based on vanadate sensitivity. The initial rate of fluorescence quenching (V₀) was calculated from the initial slope of fluorescence decline, while the maximum change in fluorescence (∆MF) was determined using the formula ∆MF = (Feq/Fmax) × 100, where Feq represents the stabilized fluorescence and Fmax the maximum initial fluorescence. Coupling efficiency was estimated by the ratio of proton pumping V₀ to the specific ATP hydrolysis rate measured at each germination stage.

2.4. Phenotypic Characterization of the Embryonic Axis During Germination

Soybean seeds were germinated as described in plant material. Two biological replicas with 15 seeds per plate were germinated with the hormones indole-3-acetic acid (IAA) (I-2886; Sigma-Aldrich, St. Louis, MO, USA), cis-abscisic acid (ABA) (A-1049; Sigma-Aldrich, St. Louis, MO, USA), 2,4D (CDS005520; Sigma-Aldrich, St. Louis, MO, USA), and the ATPase inhibitor—VAD (Na₃VO₄; Sigma-Aldrich) diluted in distilled water. Hormones and the orthovanadate were at 10 µM [20] and 100 µM final concentrations [21], respectively. To measure the senso stricto germination, the radicle protrusion was analyzed in seeds at 15, 18, 21 and 24 HAI. Embryonic axes were collected at 24 HAI. The area of embryonic axes was measured using the ImageJ version 1.54p (https://imagej.nih.gov/euj/, accessed on 6 July 2025) and their fresh weight was taken.

2.5. Bromocresol Purple Assay

To verify the extracellular acidification by the H+-ATPase proton pumping, a bromocresol assay was carried out. The BRS 284 soybean seeds were treated for 30 min with the hormones IAA (I-2886; Sigma-Aldrich, St. Louis, MO, USA), ABA (A-1049; Sigma-Aldrich, St. Louis, MO, USA), 2,4D (CDS005520; Sigma-Aldrich, St. Louis, MO, USA), and the ATPase inhibitor (VAD—Na₃VO₄; Sigma-Aldrich, St. Louis, MO, USA). Hormones and orthovanadate were at 10 µM and 100 µM final concentrations, respectively, in a 12 mL in a Petri dish (9 cm × 9 cm). Two biological replicas with 15 seeds per plate were treated. After the indicated time, the seeds were transferred to the Petri dish (15 cm × 15 cm) with 25 mL of bromocresol purple solution. For the preparation of the bromocresol purple solution, we added 1% of agar and 0.01 g of bromocresol purple in 100 mL of distilled water [21]. The pH solution was adjusted to 6.8 with 100 mM of Tris and autoclaved for 20 min. The indicator (bromocresol purple) changes color from purple to yellow when pH is below 6.0. After the preparation of the medium, the seeds were analyzed at 3 and 24 HAI.

3. Results and Discussion

3.1. Global Expression Analysis of Expansin Genes in Embryonic Axis During Germination

During germination, several physiological processes occur in seeds to support the radicle protrusion. Some of these are related to expansin genes that play a crucial role in this stage of development [8,22]. Expansins consist of a superfamily of proteins responsible for the cell wall loosening. Four subfamilies are described: α-expansin (EXPA), β-expansin (EXPB), expansin-like A (EXLA) and expansin-like B (EXLB) [23]. These proteins influence plant cell expansion, a phenomenon related to increased turgor pressure and decreased pH [10,24]. According to Zhu et al. (2014) [23], 75 expansin genes from these four classes were identified in the soybean genome. To investigate the participation of expansin members during soybean seed germination, we analyzed the expression of all 75 expansin genes in the soybean germinating embryonic axis transcriptome (Figure 1).

Among the soybean expansins, 42 genes were expressed in embryonic axes during germination (TPM > 1). Expansin A represented 81% of the expressed genes. This expasin subfamily has been related to cell wall movement acting on cellulose microfibrils [22]. We identified a group of expansins with increased expression at different points of germination. This was represented by 19 genes, 16 of them from the GmEXPA subfamily, 2 GmEXLB and 1 GmEXLA gene. From these, we can highlight GmEXPA40, GmEXPA20, and GmEXLA1, which were more expressed at 3 and 24 HAI (Figure 1a), suggesting they could have an important role in germination. The other genes of the subfamilies GmEXLA and GmEXLB showed low expression throughout the germination. One expansin gene—GmEXPA37—showed the greatest expression among expansins throughout germination (Figure 1a,b). This gene was reported by Sangi et al., 2019 and Montechiarini et al., 2020 [8,9], being essentially linked to increased cell expansion. In this last study, the GmEXPA37 was found to be expressed in the elongation zone of the embryonic axis during germination and downregulated by ABA. The increased expression of the GmEXPA37 during soybean germination was corroborated by our qRT-PCR analysis (Figure 1b).

3.2. Expression and Activity of H+-ATPase in Embryonic Axis During Soybean Seed Germination

The plasma membrane proton pump (PM H+-ATPase) plays an important role in the transport of ions, which may function as essential nutrients for various physiological processes [25]. The main function of this pump is to generate an H+ electrochemical gradient, providing a driving force for the influx and efflux of ions and metabolites across the plasma membrane. The pumping of H+ results in acidification of the cell wall, facilitating cell expansion [26]. Cellular expansion mediated by acidification of the cell wall increases their extensibility, activating several proteins, such as expansins [27]. To verify the possible role of PM H+-ATPases in embryonic axis elongation, we first analyzed their expression profile throughout the soybean germination using transcriptome data from the Soybean Expression Atlas [15]. Based on the previous annotation of H+-ATPases in soybean [28], we verified in the Soybean Expression Atlas the expression of 24 genes (Supplementary Table S1). Figure 2 shows that the H+-ATPase genes GmHA4, GmHA5, GmHA6, GmHA7, GmHA11, GmHA12, GmHA13, and GmHA21 showed detectable expression in embryonic axis during germination. Most of the genes increased their expression between 12 and 24 HAI, with the exception of GmHA12. The GmHA6, GmHA7, GmHA11, and GmHA12 genes have already been found to be highly expressed in seeds [28]. The GmHA4, GmHA5, and GmHA21 genes were differentially expressed in embryonic axes at 6 HAI (Figure 2), something which could be associated with the activation of acid growth.

To verify the activity of the PM H⁺-ATPase in embryonic axes during germination, we analyzed the specific ATP hydrolytic activity and proton pumping at 3, 6, 12 and 24 HAI (Figure 3a,b). The hydrolytic activity of PM H+-ATPase was detected at 3 HAI, with significant increase at 12 and 24 HAI (Figure 3a). For the H+ pumping activity, we found greater pumping of protons in 24 HAI than other germination points (Figure 3b). The efficiency of PM H+-ATPase pumps was also verified by the coupling ratio. The Supplementary Table S2 shows the V0 of H+ pumping and ∆MF of each germination point. The coupling was calculated by the ratio between V0 of H+ pumping and specific ATP hydrolysis. The PM H+-ATPase pumps were less efficient at 24 HAI (Figure 3c), suggesting an important role of this type of H+-ATPase in the beginning of germination.

3.3. Signaling Cascade of Auxin/ABA-Mediated H+-ATPase Regulation

Studies have shown that plant hormones play an important role in proton efflux, to acidify the apoplast, facilitating the absorption of solutes and water and promoting cell expansion [10,29]. Regarding the H+-ATPase regulators, the plant hormones auxin and abscisic acid stand out. In certain developmental stages, these play an antagonist role in the regulation of acid growth, once they can induce a signaling cascade of activation or inhibition of H+-ATPases, respectively [10,13,14]. Thus, we investigated the expression of genes involved in the auxin/ABA-mediated signaling pathway regulating H+-ATPases (Figure 4) (Supplementary Table S3).

In Figure 4, we show the signaling cascade of H+-ATPase mediated by auxin. Initially, we can highlight the influx-carrying protein family AUXIN1/LIKE-AUX1 (AUX1/LAX), whose genes presented increased expression at 6 to 12 HAI. This expression profile suggests that the activation of H+-ATPase pathway is performed by endogenous auxin on embryonic axis cells. The differential expression of two PIN-FORMED (PIN) genes in the beginning of germination—3 HAI—corroborates this profile (Supplementary Table S3). The PIN family plays a crucial role in the directionality of the auxin efflux from the cell [30]. The PIN2 could help the embryonic axis to withdraw the auxin stored during seed development, as auxin is involved in the maintenance of seed dormancy through activation of ABA signaling [6].

The embryonic axis endogenous auxin could activate the TIR1/AFB complex in the first hours of germination. We found a significant increase of expression of the AUXIN SIGNALING F-BOX 3 (AFB3) at 3 HAI (Figure 4 and Supplementary Table S3). The activation of AFB repressed the auxin-induced Aux/IAA. From 38 AUX/IAA genes, 27 showed no significant change in the expression at 3 HAI (Supplementary Table S3). Among the differentially expressed genes (DEGs) at 3 HAI, the SHY2/IAA3 showed remarkable expression throughout the germination, which could help the blocking of auxin-signaling to allow soybean germination. The SHY2/IAA3 are reported to be rapidly induced by auxin and downregulate other Aux/IAA, GRETCHEN HAGEN 3 (GH3), and SMALL AUXIN-UP RNA (SAUR) [31]. Thus, we suggest that there must be a certain specificity of the Aux/IAA family and the activation pathway of H+-ATPase.

Following the auxin signaling pathway, we found the AUXIN RESPONSE FACTORs 16 and 18 (ARF16 and ARF18, respectively) with significant increase of expression at 3 HAI and 12 HAI, respectively (Figure 4). Although ARF16 is correlated with seed dormancy by controlling ABSCISIC ACID INSENSITIVE 3 (ABI3), there are no data about ARF18 targets. Thus, the significant induction of SAUR6 and GH3 at 3 HAI (Figure 4 and Supplementary Table S3) could be due to the direct action of SHY2/IAA3, once this Aux/IAA member induces SAUR1, a sister group in the SAUR Clade I [32]. The Clade I of SAUR genes contains the SAUR19-subclade [31], which has been correlated to the H+-ATPase activation through inhibition of PP2C-D [10]. The significant increase of the auxin homeostasis gene—GH3—at 3 HAI, supports the presence of auxin in embryonic axis cells in the beginning of germination.

In the center of the auxin-mediated activation of H+-ATPase is the phosphatase type 2C subfamily D (PP2C-D). As shown in Figure 4, three genes of PP2C-D were regulated during soybean germination. The PP2C-D5 ortholog was upregulated at 3 HAI and two PP2C-D9 orthologs were upregulated at 24 HAI. These members of PP2C-D subfamily are linked to H+-ATPase-mediated cell expansion [10]. The inhibition of these PP2C-D by SAUR will lead to a phosphorylated H+-ATPase, promoting the pumping of protons and consequent cell expansion.

Regarding the auxin biosynthesis pathway, we found DEGs only at the end of germination. For example, the genes YUCCA (YUC) and TRYPTOPHAN AMINOTRANSPHERASE OF ARABIDOPSIS (TAA), which play an important role in this path [33]. The YUC8 gene was differentially expressed only at 24 HAI. However, no TAA was differentially expressed, suggesting low or no auxin biosynthesis in the embryonic axis. The activation of the auxin biosynthesis pathway at the end of germination suggests that the auxin necessary for the gravitropism and phototropism in soybean hypocotyls [34] can return to the embryonic axis cells at the end of germination by passive diffusion after acidification of apoplast. In the low pH of apoplasts promoted by H+-ATPase pumping, the auxin molecules stay protonated and can pass directly through the cell membrane [30].

In Figure 4, we show the action of the ABA pathway involved in H+-ATPase regulation. This hormone is responsible for transmitting a signal to a soluble receptor pathway PYRABACTIN RESISTANCE 1/PYR1-LIKE/REGULATORY COMPONENT OF ABA RECEPTOR (PYR/PYL/RCAR) which inhibits the catalytic activity of PP2C subfamily A (PP2C-A), keeping the SUCROSE NON-FERMENTING-1 (SNF1)-RELATED protein kinase 2 (SnRK2) phosphorylated [35]. We found three PYR-like genes with significant increase of expression at 3 HAI and decreased expression during germination (Figure 4 and Supplementary Table S3), suggesting that the ABA is present in the soybean embryonic axes at the beginning of germination and decreases as germination proceeds. The expression of six PP2C-A genes is low during germination, being significantly increased at 24 HAI (Figure 4). Regarding SnRK2, three genes were expressed (SnRK 2.10, 2.13 and 2.20). According to Zhao et al. (2017) [36] the soybean SnRK2.10 and SnRK2.13 are in the same group as SnRK2.2 and SnRK2.3 from Arabidopsis, respectively. The SnRK2.2 and SnRK2.3 are required in the control of ABA responses in Arabidopsis seed germination [37]. Planes et al. (2015) [13] have shown that SnRK2.2 and SnRK2.3 are activated in the presence of ABA and phosphorylate an unidentified residue on the C-terminal of AHA2, leading to the inhibition of the H+-ATPase. In soybean embryonic axes, the expression of SnRK2.2 increased significantly only at 24 HAI (Figure 4), suggesting that the SnRK2-mediated inhibition of H+-ATPase is decreased as ABA-signaling is repressed.

3.4. Action of Auxin and ABA in the Functioning of H+-ATPase During Soybean Seed Germination

To evaluate the effect of exogenous IAA and ABA in the functioning of H+-ATPase during germination, we first analyzed the percentage of germination in five different treatments (Figure 5). Control seeds have the same germination capability as seeds germinated in IAA at 24 HAI, although the seeds germinated in medium with IAA present a delay in germination compared with the control, corroborating the study that exogenous IAA delays soybean seed germination [20]. Recent studies demonstrate that exogenous auxin induces seed dormancy, thus delaying the cell expansion, the rupture of the soybean seed coat and the protrusion of the radicle. In this sense, auxin inhibits gibberellin (GA) biosynthesis and stimulates abscisic acid (ABA) synthesis [20,38]. Seeds germinated in 2,4D medium presented a similar profile of abscisic acid throughout germination. 2,4D is an herbicide that has an auxin activity and is therefore used as a synthetic analogue of the same [39]. The orthovanadate curve shows a similar profile to that of IAA throughout germination, despite leading, at 24 HAI, to 33% less germinated seeds. Orthovanadate acts as H+-ATPase inhibitor [40], therefore negatively impacting soybean germination.

We also performed a phenotypic analysis of the embryonic axis at 24 HAI in the five different treatments (Figure 6). Sangi et al. (2019) [8], observed that the size of embryonic axes increased after 12 HAI, with the largest size found in 24 HAI when germination is complete for 93% of seeds. Embryonic axes germinated in IAA do not show significant difference in fresh mass when compared with control and orthovanadate (Figure 6a). However, exogenous IAA downsized the embryonic axis (Figure 6b). This corroborates the data indicating that exogenous auxin inhibits acid growth and nutrient mobilization [41,42]. A similar profile was found for orthovanadate-treated seeds (Figure 6). Embryonic axes germinated in 2,4D and ABA showed significant decreases of fresh mass and area, reinforcing the percentage of germination in Figure 5 and suggesting that they play a similar role in inhibiting cell expansion by H+-ATPase.

To elucidate the impact of auxin and ABA treatments in the functioning of H+-ATPase, we germinated treated seeds in the medium with bromocresol purple, where the pumping of H+ ions become visible (Figure 7). We observed that the different treatments have a similar effect on H+-ATPase, due to the formation of a purple halo (basic pH) around the seed, provided by the influx of protons in the initial hours of germination—3 HAI [42]. At the end of germination, we found that the control seed showed a yellow halo around the embryonic axis (Figure 7). This suggests that at the end of germination there was an efflux of protons and consecutive acidification of the medium by the functioning of H+-ATPase [42]. The IAA-treated seeds showed a prominent axis at 24 HAI, although with smaller size than control, as indicated in Figure 6. Shuai et al. (2017) [20] have shown that the exogenous IAA treatment of soybean seeds increased ABA concentration while decreasing GA levels at the beginning of germination (6 HAI). As shown on Figure 5, the IAA-treated seeds increased their germination after 18 HAI, suggesting that the IAA induction of ABA biosynthesis occurs in the beginning of germination and that the ABA levels decrease at some point of seed imbibition. The decrease of ABA levels could lead to inhibition of SnRK2.2/2.3 by active PP2C-A, which prevents inhibitory phosphorylation of the H+-ATPase, stimulating the acid growth of embryonic axes. The seeds treated with 2,4D showed a greater effect on H+-ATPase functioning and consequently a higher delay of germination than IAA. This profile has also been reported by Shuai et al. (2017) [20]. The treatments with ABA and orthovanadate did not show the formation of the yellow halo in the seeds and there was no protrusion of the embryonic axis at 24 HAI (Figure 7), confirming that the embryonic axis expansion is impacted by H+-ATPase and its regulators—auxin and ABA.

4. Conclusions

In this study, we identified that α-expansins are majorly regulated in soybean embryonic axes during germination. Among 42 expansins, the GmEXPA37 was the most expressed gene during soybean germination, corroborating previous works [8,9]. Three PM H+-ATPases were upregulated in embryonic axes at 6, 12, and 24 HAI. The maximum efficiency of PM H+-ATPases was at the beginning of germination, although major ATP hydrolysis and H+ pumping were detected at 24 HAI. The auxin and ABA signaling cascades involved in H+-ATPase regulation were characterized in soybean embryonic axes during germination. This analysis suggests that in the first hours of germination endogenous auxin could activate SHY2/IAA3, which may possibly regulate SAUR to inhibit PP2C-D, keeping the H+-ATPase phosphorylated and active. On the other hand, the inhibition of H+-ATPase by the pathway ABA->PYL->PP2C-A->SnRK2 could decrease as ABA-signaling is repressed throughout germination. We characterized the effect of exogenous IAA treatment on soybean seeds and found that it delayed germination, whereas 2,4D ABA and VAD treatments blocked germination. In our analysis, acid growth appears to take place during the mid-to-late stages of germination, which is corroborated with the increased expression levels of expansins. Furthermore, the sensu stricto germination blockage induced by 2,4-D, ABA, and VAD is possibly due to interference with H+-ATPase activity involved in nutrient transport, which is initiated at the early stages of the germination process [43]. We therefore conclude that H+-ATPase activity plays an important role in soybean germination, though the contribution of acid growth to this process appears to be relatively modest. Together, our results unveil an additional layer of the complex regulation of seed germination and pave the way for the finding of new controllers of soybean seed germination.