Early carbon mobilization and radicle protrusion in maize germination

Abstract

Considerable amounts of information is available on the complex carbohydrates that are mobilized and utilized by the seed to support early seedling development. These events occur after radicle has protruded from the seed. However, scarce information is available on the role of the endogenous soluble carbohydrates from the embryo in the first hours of germination. The present work analysed how the soluble carbohydrate reserves in isolated maize embryos are mobilized during 6–24 h of water imbibition, an interval that exclusively embraces the first two phases of the germination process. It was found that sucrose constitutes a very significant reserve in the scutellum and that it is efficiently consumed during the time in which the adjacent embryo axis is engaged in an active metabolism. Sucrose transporter was immunolocalized in the scutellum and in vascular elements. In parallel, a cell-wall invertase activity, which hydrolyses sucrose, developed in the embryo axis, which favoured higher glucose uptake. Sucrose and hexose transporters were active in the embryo tissues, together with the plasma membrane H⁺-ATPase, which was localized in all embryo regions involved in both nutrient transport and active cell elongation to support radicle extension. It is proposed that, during the initial maize germination phases, a net flow of sucrose takes place from the scutellum towards the embryo axis and regions that undergo elongation. During radicle extension, sucrose and hexose transporters, as well as H⁺-ATPase, become the fundamental proteins that orchestrate the transport of nutrients required for successful germination.

Introduction

Germination is a key process that allows the seed embryo to growth and to evolve into a photosynthetic organism. This process starts with the rehydration of the quiescent seed and ends with the onset of elongation of the embryo axis. This is physically visualized as radicle emergence from the seed (Mayer and Poljakoff-Mayber, 1989; Bewley and Black, 1994; Bove et al., 2002). Physical and biochemical events underlie this process: weakening of seed covers, turning on of metabolic activity, activation of gene transcription, relaxation of the embryonic cell walls, and reassembly and biogenesis of organelles, among others (Logan et al., 2001).

One of the most studied processes on seedling development is the mobilization of complex polymers such as starch, proteins, and lipids from storage tissues such as endosperm or cotyledons. These compounds, considered as seed reserves, are used as energy sources and building blocks for seedling growth in a process that has been generically designated as germination (Srivastava, 2002). However, the utilization and transport of reserves occur as two post-germination events, since they take place after radicle emergence has proceeded and they are regulated by phytohormones such as gibberellic acid and abscisic acid (Pritchard et al., 2002; Srivastava, 2002; Eastmond, 2006).

In this context, the contribution of the intrinsic carbon reserves of the embryo to sustain the discrete, but required metabolic activity that occurs prior to and at the point of radicle emergence, has been underscored. These nutrient reserves are significant (Bewley and Black, 1994; Black et al., 1996; Dierking and Bilyeu, 2009) and their close accessibility to the embryo axis makes them an especially advantageous supply of metabolic energy in terms of time and space. In fact, despite of the impressive amount of information on the physiology of germination (Mayer and Poljakoff-Mayber, 1989; Bewley and Black, 1994) and of the advances in understanding its molecular basis (Bove et al., 2002; Dekkers and Smeekens, 2007; Yang et al., 2007; Hynek et al., 2009), two central events preceding radicle emergence remain unclear: (a) the identification of the carbon source that the embryo uses to support the metabolic activity and radicle emergence; and (b) the mechanism of radicle elongation. These two events are tightly linked and confined to the first hours of seed imbibition.

The metabolic activity of the embryo is moderate but essential during the first hours of water uptake (Srivastava, 2002), since it is directed to provide the energy and precursors for the synthesis of cell components and to sustain radicle emergence, the hallmark of germination. It has been described that radicle extension involves cell division and/or cell elongation (Antipova et al., 2003), which demands the incorporation of molecules that must be de novo synthesized and the transport of nutrients from the outside to the embryo axis cells. This traffic is carried out by transmembrane carriers that are energy dependent, such as the dipeptide and tripeptide transporters which, in the barley scutellum, contribute to supply nitrogen and carbon to the embryo during the first hours of germination (Waterworth et al., 2000; Stacey et al., 2006).

In coleoptiles, whose growth requires cell elongation rather than cell division, cell enlargement is promoted by the cell-wall relaxation, which is facilitated by acidification of the apoplast (Hager, 2003; Kutschera, 2004). The low pH contributes to increase the activity of several enzymes that modify the cell wall such as expansins, endoglucanases, and xyloglucan endo-transglycosylases (Cosgrove, 2005). Experimental evidence indicates that the plasma membrane H⁺-ATPase generates such acidification (Hager, 2003; Rober-Kleber et al., 2003; Cosgrove, 2005). This enzyme catalyses the ATP hydrolysis coupled to the translocation of H⁺ from the cytosol into the apoplast, thus building a transmembrane pH difference or ΔpH (acidic outside the cell) and a difference in electrical potential or ΔΨ (negative inside the cell) (Sondergaard et al., 2004). The role of the plasma membrane H⁺-ATPase to support cell elongation in established seedlings is sustained by the correlation found between the presence or the activity (Frias et al., 1996) of this enzyme and the growing rates of coleoptiles and roots. However, the mechanism of cell elongation in seed germination remains unknown, although it is expected that radicles extending from the seed embryos follow the mechanism based on the acid-growth theory in order to develop root emergence from the seed. In fact, H⁺ secretion into the apoplast has been considered as an important condition for seed germination (Winch and Pritchard, 1999; Antipova et al., 2003; Hager, 2003). Although the presence of H⁺-ATPase in the first 5 h of seed imbibition has been documented (Sánchez-Nieto et al., 1998; Enríquez-Arredondo et al., 2005), direct experimental evidence of the role of this enzyme during the time span in which radicle emergence takes place is not available.

The present work analysed the endogenous sugar mobilization between maize scutella and embryo axes and the function of three plasma membrane transporters during the germination hours (6–24 h), in which both tissues are involved in the onset and accomplishment of radicle emergence. The results suggest that endogenous sucrose from the scutellum flows towards the embryo axis to support the cell elongation mainly at the radicle region, and that sucrose and hexose transporters as well as H⁺-ATPase are fundamental proteins to orchestrate this transport in order to lead the embryo to culminate germination by radicle extension.

Materials and methods

Biological material and imbibition conditions

Embryos contain the embryo axis plus the scutellum. Therefore, the contribution to germination by these two tissues was studied in isolated embryos and dissected embryo axes upon scutella removal. Embryos were manually dissected from mature, dry maize (Zea mays Landrace Chalqueño) seeds, thoroughly removing the endosperm with a scalpel. Embryo axes were removed from the embryos with a scalpel and stored at –4 °C until use.

Embryos and embryo axes were disinfected with 0.12% (v/v) sodium hypochloride just before imbibition. This was done placing embryos or embryo axes in 1% agar in Petri dishes and incubating at 29 °C at different times under sterile and dark conditions. When the imbibition period was completed, intact embryos or embryo axes were immediately used to determine physiological parameters. When biochemical or membrane isolation was performed, embryos or embryo axes were immediately frozen in liquid nitrogen and maintained at –70 °C until use. When required for biochemical analyses, embryos or embryo axes were ground in a mortar with liquid nitrogen until a fine powder was obtained.

Determination of germination, water uptake, and radicle elongation

An embryo was considered germinated when its radicle reached 3 mm. Active water uptake by embryos or embryo axes was monitored by the increase in fresh weight during imbibition time. Radicle elongation was determined by measuring its length with a Vernier microscope.

Determination of oxygen uptake

Oxygen consumption by maize embryos or embryo axes was determined polarographically with a Clark type oxygen electrode. Once imbibition time ended, maize embryos or embryo axes were added to 5 ml water under constant stirring with a magnetic bar. Oxygen uptake was recorded during the initial 4 min.

Determination of medium acidification

Thirty embryos imbibed at the indicated times were added into 10 ml of 250 mM sucrose and 2 mM MES-TEA (pH 6.0). The pH electrode was previously equilibrated in this solution at 30 °C. When embryos were added, electrode potentials were continuously measured for 15–20 min, as described in Gutiérrez-Nájera et al. (2005).

Determination of carbohydrates

Embryo extracts were used to measure soluble carbohydrates. Sucrose, glucose, and fructose were determined by enzymic coupled assays. For soluble sugar determinations, 200 mg powdered embryo or embryo axis was extracted twice by grinding with 5 ml of 80% ethanol at 80 °C. The liquid phase was centrifuged at 1300 g for 10 min. The supernatants of the two extracts were combined, heated at 70 °C, and evaporated to dryness. Residues were reconstituted with 300 μl deionized water. The concentrations of glucose, fructose, and sucrose were determined using an enzymic assay coupled to NAD production and glucose assay reagent (GAR, Sigma-Aldrich, St Louis, MO, USA) with some modifications. In brief, 1–12 μl sample was loaded in a 96-microwell plate, followed by 200 μl GAR; the reaction was allowed to proceed for 20 min at 25 °C. The free forms of glucose and fructose in the sample were phosphorylated by hexokinase to yield glucose-6P and fructose-6P. Subsequently, with glucose-6P dehydrogenase and NAD⁺, glucose-6P was detected by NADH absorbance at 340 nm. Fructose-6P was converted to glucose-6P by adding 2 μl of 1.2 U/ml phosphoglucose isomerase, mixing and incubation for 5 min at 25 °C before measurement of NAD⁺ reduction. Sucrose was subjected to enzymic hydrolysis under acid conditions to produce fructose and glucose by mixing 12 μl sample and 4 μl of 80 mg/ml invertase (Sigma-Aldrich) in 200 mM magnesium acetate (pH 5.0) followed by 2 h incubation at 37 °C. Then, the released glucose and fructose were quantified as described above. The produced NADH was measured by absorbance at 340 nm using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

Determination of triglycerides

Lipid extraction was performed as described by Folch et al. (1957) with some modifications and separated by thin layer chromatography, as described in Supplementary Materials and Methods (available at JXB online).

Determination of enzyme activities

Cell-wall invertase extracts were obtained essentially as reported by Pelleschi et al. (1997). Then, activity was determined by an enzymic assay coupled to NADPH absorbance, as described by Bergmeyer and Bemt (1974). The hexokinase activity was measured as described by Dai et al. (1999) using the soluble fraction from the homogenized embryo tissues. H⁺-ATPase activity was measured by spectrophotometric quantification of inorganic phosphate release, according to González-Romo et al. (1992), as detailed in Supplementary Materials and Methods.

Determination of sucrose and glucose uptake

Sucrose and glucose uptake by isolated embryos were measured by an isotopic assay. For sucrose uptake, isolated embryos were incubated at 25 °C in a transport buffer containing 50 mM HEPES-HCl (pH 6.0) 330 mM sorbitol, 1 μCi [¹⁴C]-sucrose (410 mCi mmol⁻¹, Perkin Elmer Life Science, Boston, MA, USA), and 10 mM sucrose. Glucose uptake was measured by sucrose substitution in the transport buffer using 1 μCi [¹⁴C]-glucose (310 mCi mmol⁻¹, Perkin Elmer Life Science) and 10 mM glucose. After 10 min incubation, embryos were washed twice with 2.5 ml transport buffer containing a 10-fold excess of non-radioactive sucrose or glucose, depending on the labelled sugar. Subsequently, embryos were air-dried on filter paper and, to release the radioactive sugar, embryos were placed in a microtube containing 500 μl lysis medium (37% H²O²/concentated HClO⁴, 2:1, v/v) for 5 h with occasional mixing. The final solution (300 μl) was added to scintillation liquid (3 ml) and radioactivity was detected using a multipurpose scintillation counter (LS 6500, Beckman Coulter, Brea, CA, USA).

Determination of transmembrane ΔpH

This was performed by the measurement of the quenching of 9-amine-6-chloro-2-methoxyacridine (ACMA) fluorescence, as described in Supplementary Materials and Methods.

Preparation of liposomes

Asolectin phospholipids were used to obtain liposomes by sonication, as described in Supplementary Materials and Methods.

Determination of protein

Protein determination was done using a modification of the method of Lowry et al. (Peterson, 1977).

Isolation of microsomal and plasma membrane vesicles

Total membranes and plasma membrane vesicles (PMV) were obtained by differential centrifugation and two-phase partitioning in aqueous polymers, respectively, as detailed in Supplementary Materials and Methods.

Antibody preparation

Polyclonal antibodies against antigenic sequences from the sucrose and hexose transporters were prepared in rabbit using the procedure of González-Halphen et al. (1988), as described in Supplementary Materials and Methods and Supplementary Fig. S1.

Electrophoresis and immunodetection by Western blot

Microsomal proteins were separated in SDS-PAGE gels and then electrotransferred to PVDF membranes, as detailed in Supplementary Materials and Methods.

Histochemical localization of sucrose

Embryo fixation was essentially performed as reported by Baud and Graham (2006). Briefly, embryos from every imbibition time were placed in a microtube containing 1 ml of 2% paraformaldehyde, 2% polyvinylpyrrolidone-40, and 1 mM DTT and left for 1 h at 4 °C. Then, they were washed five times with 1 ml deionized water and stored overnight at 4 °C. In situ detection of sucrose was carried out by hydrolysis with a commercial invertase (Sigma-Aldrich) and then measuring the released glucose in the embryos by an enzymic method. The latter couples the hexokinase and glucose phosphate dehydrogenase activities to NADH formation, which is used for nitroblue tetrazolium reduction, thus staining the sucrose-containing tissues in dark blue.

Immunohistochemistry of the plasma membrane H⁺-ATPase and the sucrose transporter

Sucrose transporter and H⁺-ATPase localization was carried out in embryo tissue sections using specific antibodies. Briefly, embryos imbibed for 24 h were fixed in 4% p-formaldehyde at pH 6.9 for 1 h, washed three times. and infiltrated in 320 mM sucrose for 24 h at 4 °C. Frozen sections (8 μm) were prepared in a cryostat and collected on gelatin-coated slides (Enríquez-Arredondo et al., 2005). Sucrose transporter localization was performed by using the respective specific antibodies, described in Supplementary Materials and Methods. H⁺-ATPase localization was made with a specific antibody directed against the amino-terminal of the maize H⁺-ATPase, as described in Enríquez-Arredondo et al. (2005). Endogenous peroxidase activity was removed from tissue sections by treating the tissues with 2.5% H₂O₂ and 1% periodic acid during 1 h, or by incubating with 0.3% H₂O₂ in methanol. Non-specific binding of immunoglobulin was blocked with 2.5% non-fat skimmed milk with 1:60 rat serum in blocking PBS [10 mM phosphate buffer (pH 7.4), 150 mM NaCl, 2% cellulase, 5 mM MgSO₄, 0.01% Triton X-100, 5 mM EDTA, and 0.01% polyvinylpolypyrrolidone). After three washes of 5 min, sections were incubated with the primary specific antibody (1:50 for H⁺ -ATPase and 1:200 for sucrose transporter) for 72 h at 7 °C. Secondary antibody conjugated to horseradish peroxidase was diluted 1:50 in PBS and incubated 1 h at room temperature. After three washes with PBS, the complex was detected by addition of 3-amino-9-ethylcarbazole [40 mg in 100 ml of acetate buffer (pH 5.0)] in the presence of H₂O₂ (25 μl of fresh 30% hydrogen peroxide). Controls were treated with pre-immune serum or omitting the specific antibody (Enríquez-Arredondo et al., 2005). Nomarski differential interference contrast microscopy in an Axoskop Zeiss microscope was utilized for visualization and photography.

Results and discussion

Physiological parameters during maize embryo germination

In addition to the aleurone layer, the scutellum and the embryo axis include the live tissues of the seed and the molecular entities involved in reserve mobilization (Mayer and Poljakoff-Mayber, 1989). The scutellum and the embryo axis can be considered as source and sink compartments, respectively, in the same way that a mature plant synthesizes and translocates sucrose from source to sink tissues or organs. In the present work, whole embryos and embryo axes were used to explore whether their endogenous soluble carbohydrates were mobilized at the early times of seed imbibition and to identify the molecular paths involved in this process. Participation of the endosperm reserve mobilization was not considered since this is a post-germination event (Srivastava, 2002). Therefore, germination in the embryo and the embryo axis was first characterized according to several physiological parameters during the first 40 h in water imbibition. These parameters were: germination rate, water and oxygen uptakes, and radicle elongation.

Germination

Maize embryos showed a maximal germination of 97% at 24 h (Fig. 1A). Since germination was carried without external supply of sugar, this result indicated that embryos contained enough reserves to perform a fast and successful germination. As a reference, the germination rate of whole maize seeds following imbibition was also undertaken. Seeds reached similar maximal germination than embryos (Fig. 1A) but after longer times (60 h), presumably due to the presence of the testa and the pericarp in the seeds, covers that are removed when embryos are dissected. Both the pericarp and the testa function as permeability barriers to the diffusion of oxygen and as mechanical barriers impeding radicle protrusion (Miyoshi and Sato, 1997).

Fig. 1.

Physiological parameters during maize embryo imbibition. (A) Time course of germination during embryo or seed imbibition. (B) Time course of water and oxygen uptake measured as increase in fresh weight and oxygen consumption in embryos, respectively. (C) Time course of radicle elongation in embryos during imbibition; inset shows radicle elongation velocity calculated as the first derivative of the non-linear fit from the line in the main graph. Results are representative of at least three independent replicates.

Water uptake

It is considered that seed germination concludes with the radicle protrusion, which occurs towards the end of phase II of water uptake by whole seeds (Bewley and Black, 1994). Therefore, germination sensu stricto embraces only phases I and II of seed hydration (Bewley and Black, 1994). In order to allocate this period in maize embryos, the kinetics of water uptake were followed (Fig. 1B). The classical three-phase water uptake behaviour was observed: phase 1, characterized by fast water uptake due to the hydration of seed materials, followed by phase II, wherein a discrete level of metabolic activity emerges and the water content remains constant. This phase ends with the growth of the embryo axis that is manifested in its terminal portions, the plumule and the radicle. The latter extends from the surrounding tissues, breaking up the seminal cover and emerging from the seed (Bewley and Black, 1994). In the present work, the transition between phases II and III was estimated at about 18 h, which was near the time of maximal germination (24 h).

Oxygen consumption

The rate of oxygen uptake was significant after 8 h of imbibition and then slowly increased, attaining higher rates once the radicle had emerged and active seedling growth was in progress (Fig. 1B). This is consistent with the appearance of mitochondrial respiratory and phosphorylating activities in germinating maize embryos, as early as 1 or 6 h, depending on imbibition conditions (Logan et al., 2001). Oxygen uptake was an important indication that metabolic activity was being carried out by the embryos in these conditions, since the mitochondrial function greatly depends on active protein synthesis and gene transcription (Logan et al., 2001). In addition, functional mitochondria contribute to provide adequate levels of ATP and metabolic precursors for the anabolic activities of phases II and III.

Radicle growth

Embryos showed exponential rates of radicle extension (Fig. 1C) reaching a maximal rate at about 16 h, as indicated by deriving the equation of the experimental curve (Fig. 1C, inset). It was observed that radicle growth rate diminished with a trend to zero at longer times. This behaviour suggests that an active process of cell elongation takes place in this time span in order to support radicle extension, since it has been reported that cell division does not contribute to this step during maize germination (Antipova et al., 2003).

The characterization of the aforementioned physiological parameters defined a window time of 6–24 h in which the embryos are competent to complete the germination programme. This allowed the study of the role of the endogenous embryo reserves and of some of the key transporters involved.

Soluble carbohydrate levels

Analysis of the main soluble carbohydrates indicated that monosaccharides such as glucose and fructose were in very low amounts both in embryos and embryo axes and showed negligible variations along the first 24 h of water imbibition (Table 1). On the contrary, sucrose was remarkably high as compared to glucose and fructose, both in the embryo and the embryo axis. Specifically, sucrose content at 0 h was 10-fold higher in the embryo as compared to the embryo axis. This difference increased 17-fold at 12 h. This suggested that during the first 12 h, sucrose was rapidly mobilized within the embryo and used by the embryo axis (Table 1). The sucrose remaining in both tissues after 24 h was about 16% of the original reserves (Table 1). The large amounts of sucrose that diminished in the embryo throughout these 24 h were apparently exported by the scutellum towards the embryo axis. Here, it is worth recalling that the scutellum does not undergo growth and its main function is to provide carbon and nitrogen backbones to the embryo axis, which in turn does not have enough reserve molecules and is engaged in a programme of active growth in germination (Mayer and Poljakoff-Mayber, 1989). It is possible that the scutellum protein and lipid reserves, and to minor extent its own sucrose, may become a source of energy for sugar transport to the embryo axis (this study; Wang and Huang, 1987; Salmenkallio and Sopanen, 1989).

Table 1.

Soluble carbohydrate levels in the embryo and the embryo axis during germination

Imbibition time (h)	Carbohydrate per embryo axis (mg)			Carbohydrate per embryo (mg)
	Sucrose	Glucose	Fructose	Sucrose	Glucose	Fructose
0	0.945 ± 0.282	0.108 ± 0.003	0.007 ± 0.001	9.092 ± 0.217	0.0834 ± 0.002	0.006 ± 0.001
8	0.420 ± 0.064	0.046 ± 0.001	0.003 ± 0.003	5.446 ± 0.022	0.086 ± 0.034	0.025 ± 0.010
12	0.249 ± 0.023	0.075 ± 0.001	0.019 ± 0.005	4.272 ± 0.032	0.082 ± 0.050	0.015 ± 0.001
24	0.151 ± 0.014	0.039 ± 0.005	0.019 ± 0.006	1.486 ± 0.020	0.047 ± 0.005	0.034 ± 0.017

Embryo and embryo axis were germinated at the indicated times and then carbohydrates were extracted in ethanol and quantified by enzymic coupled assays, as described in Materials and Methods. Values are mean ± SD (n =12).

Although glucose, fructose, maltose, and raffinose are soluble sugars commonly found in dry seeds, they are usually present in relatively low amounts (Edelman et al., 1959; Black et al., 1996). Two lines of soybean seeds with different content of raffinose do not exhibit differences in their germination capacity (Dierking and Bilyeu, 2009), suggesting that in soybean raffinose is not necessary for supporting germination. On the contrary, sucrose is the most abundant soluble carbohydrate in maize (87–83%) (Edelman et al., 1959; Bernal-Lugo and Leopold, 1992), rice (60%) (Scofield et al., 2007a), and wheat seeds (54%) (Black et al., 1996).

Decrease in total sucrose content in the embryo and embryo axis was further established by in situ localization of sucrose at different times of germination. Initially, the scutellum showed extensive areas with high sucrose (Fig. 2A), which were exhausted after 8 h, and new areas with sucrose developed at the border between the scutellum and the embryo axis (a boundary that contains the line of cells forming the scutellum parenchyma). Later (18 and 24 h), sucrose-rich areas were found at the upper tip of the embryo axis, in the plumula region, which is starting to undergo cell growth to originate the seedling leaves. Remarkably, the radicle region was never positive to sucrose stain, while a reliable signal of sucrose content in the embryo was only apparent after 12 h, close to the time (16 h) at which maximal velocity of radicle elongation was attained (Fig. 1A). These results and the kinetics of sucrose decline (Table 1) suggested that sucrose was efficiently mobilized and consumed, probably serving as the main energy source to sustain the vigorous radicle extension of the embryo axis.

Fig. 2.

Profiles of sucrose and triglyceride contents and invertase and hexokinase activities during maize embryo germination. (A) Sucrose localization in embryos imbibed at different times; left image, schematic representation of the components of the maize seed in a longitudinal slice; other images, longitudinal views from embryos imbibed at 0, 8, 12, 18, and 24 h, as indicated. Arrows indicate sucrose-stained regions. Representative images are shown for each imbibition time from three independent experiments. p, Plumula region; r, radicle region; sc, scutellum; scp, scutellum parenchyma cells. (B) Cell-wall invertase and hexokinase activities were measured in the embryo or the embryo axis at the indicated times using 25 μg of protein in an assay coupled to the production of NADH. Values represent the corresponding average values of 12 replicates ± SD. (C) Triglyceride content was quantified in the scutellum or the embryo axis imbibed during the indicated times. Values represent the corresponding average values of nine replicates ± SD. CW, Cell wall; HK, hexokinase.

In addition to sucrose, stored proteins in the scutellum may contribute carbon, and mainly nitrogen backbones, to radicle growth. In fact, a high content of stored protein (Salmenkallio and Sopanen, 1989), elevated levels of proteinases and carboxipeptidases (Feller et al., 1978), and a high expression of oligopeptide transporters (Waterworth et al., 2000; Stacey et al., 2006) are present in barley and maize scutella during the germination phases. It is possible that the scutellum may use proteins as a source for sucrose synthesis, since this tissue expresses all the required enzymes and a poor intracellular invertase activity (Edelman et al., 1959).

The observed decay of sucrose in the scutellum (Table 1) led this study to explore the possible pathways of its utilization. Sucrose mobilization and its carbon backbone cleavage into hexoses is a very common strategy in plants to acquire metabolic energy in an efficient way (Roitsch and González, 2004; Slewinski et al., 2009). During maize embryogenesis, cell-wall invertases have an important role in the sucrose breakdown to produce hexoses that nourish the developing embryo (Doeblert and Felker, 1987). A low cell-wall invertase activity was observed in embryos and embryo axes during the first 8 h of imbibition (Fig. 2B). Then, embryo axes developed very high invertase activity that at 24 h was about 10-fold higher than the activity observed at 0 h. In contrast, embryos displayed low and constant invertase activity levels throughout this time. Considering that the weights of the embryo and the embryo axis were 0.1536 ± 0.046 g (n = 20) and 0.013 ± 0.003 g (n = 20), respectively, it was evident that the total cell-wall invertase activity in the embryo axis was significantly higher. These results advocate that the embryo axis (behaving as a sink) is fed with the hexoses originating from the action of cell-wall invertase on the sucrose mobilized from the scutellum tissue (source tissue), which contains a low invertase activity (Fig. 2B; see also Edelman et al., 1959). The key role of the cell-wall invertase in this germination step is in agreement with the high invertase activity found in sinks at different stages of plant development (Sturm and Tang, 1999).

Given the large amounts of sucrose mobilized from the scutellum, high amounts of glucose and fructose would be expected. However, very low levels of these monosaccharides were determined (Table 1), suggesting that they are rapidly metabolized, mainly through the glycolytic pathway. To test this hypothesis, hexokinase activity, the first controlling glycolytic enzyme, was assessed. Both embryos and embryo axes displayed low hexokinase specific activity up to 8 h of imbibition that later increased at 24 h (Fig. 2B). These results suggested that the glycolytic pathway was active in this time span and that sucrose could be used by the embryo axis to produce hexoses as energy source to sustain the basic metabolic activities prevailing in this period that lead to radicle protrusion.

Lipid mobilization

It has been reported that lipid stores in the seed are used as a carbon source mainly to sustain seedling growth, as shown with lipase mutants that are capable of germination but are unable to proceed to seedling development (Quettier and Eastmond, 2009). Since maize embryo tissues, particularly the scutellum, contain a high content of triglycerides (TG), the present study examined its content along the imbibition (Fig. 2C). TG gradually declined, albeit at very low rates, in both embryonic tissues during the first 12 h. Afterwards, the mobilization of TG increased to attain 12 and 15% reduction at 30 h both in the embryo and the embryo axis, respectively (Fig. 2C). Therefore, these reserves were indeed consumed, but at post-germination times (50 h) when lipase is activated (Wang and Huang, 1987). The modest amounts of TG consumed and the high TG levels remaining after germination indicated that the lipid reserve was not the main energy source to sustain germination of the embryo or the embryo axis.

Glucose and sucrose transport

To establish whether the embryos were competent for soluble carbohydrate import, sucrose and glucose uptake was examined during the first 30 h of water imbibition. Non-imbibed embryos showed ability to transport glucose, while maximal transport rate was reached after 6–30 h of imbibition (Fig. 3A). In contrast, sucrose uptake was lower and constant throughout the same interval. Isolated embryo axes were also able to transport glucose and sucrose, but at 3–5-fold lower levels between 8 and 30 h as compared to whole embryos (Fig. 3B). It has been reported that ZmSUT1 is a protein that may transport sucrose into the cell (Slewinski et al., 2009) but is able to drive the efflux of sucrose with a K _m of 3.6 mM (Carpaneto et al., 2005). Taken together, the results shown in Figs. 2 and 3 suggest that the sucrose present in the embryo scutellum can be exported (Table 1) to the symplast and from there it could be distributed to the apoplast from the embryo axis. There, sucrose could be hydrolysed by the cell-wall invertase to glucose and fructose (Fig. 2B) in order to be taken up by the embryo axis cells (Fig. 3). It is possible that some of the sucrose in the scutellum apoplast is also taken up by the scutellum cells and then hydrolysed by internal invertases (Sturm and Tang, 1999; Roitsch and González, 2004), to support their metabolic activities that lead to completion of germination.

Fig. 3.

Sucrose and glucose uptake and levels of sucrose and hexose transporters and H⁺-ATPase in the embryo and the embryo axis during imbibition. (A, B) Sucrose and glucose uptake by whole embryos (A) and embryo axes (B); embryos or embryo axes were incubated with [¹⁴C]-sucrose or [¹⁴C]-glucose in a reaction medium for 10 min at 25 °C. The labelled sugar was released after cell lysis and quantified by liquid scintillation. Values represent the corresponding average values of six independent replicates ± SD. (C) Representative Western blots showing levels of sucrose and hexose transporters and H⁺-ATPase. Proteins from microsomal fractions of embryos imbibed during the indicated times were separated by SDS-PAGE and subjected to Western analyses. Immunodetection of sucrose and hexose transporters and H⁺-ATPase were done using specific antibodies at least in three independent membrane preparations.

The way that sucrose and glucose are transported into both the scutellum and the embryo axis was explored. Sugar transport takes place via plasmodesmata through the symplast and/or by transmembrane cell movement mediated by energy-dependent sugar carrier proteins (Sauer, 2007). In the latter mechanism, sucrose and hexose transporters work in source or sink tissues: Both transporters are members of the major facilitator superfamily located at the plasma membrane (Sauer, 2007). Sucrose transporters (SUT) function as sucrose-proton symporters with a 1:1 stoichiometry that use the energy from the H⁺ gradient established by the plasma membrane H⁺-ATPase. To establish the presence of these transporters in the germination process, immunodetection assays were done in the membrane fractions from the maize embryos at 0, 6, 12, 18, and 24 h of imbibition. Hexose and sucrose transporters were present at low levels in the non-imbibed embryos, while upon imbibition, levels increased at the same extent at all times (Fig. 3C, Supplementary Fig. S2). A comparison of the transporter content profiles (Fig. 3C, Supplementary Fig. S2) with the hexose and sucrose uptake by the embryos (Fig. 3A) indicated an absence of a close correlation. However, this apparent dissimilarity could be explained by the appearance of new transporter isoforms. This is a common event that occurs in the progress of germination at early times (Andrianarison and Beneytout, 1992; Waterworth et al., 2000; Enríquez-Arredondo et al., 2005; Sánchez-Nieto et al., 2011). In the case of glucose (Fig. 3A, C), an initial isoform with basal activity could be substituted by levels of another isoform with higher activity. In the case of sucrose, the 0 h transporter, present in lower amount, could be relayed by another form that, although in larger amounts, has the same basal activity as the initial form. However, it can be acknowledged that the different forms of transporters may correspond either to different isoenzymes or to the same proteins regulated by factors that are produced in the course of imbibition (Andrianarison and Beneytout, 1992; Waterworth et al., 2000; Enríquez-Arredondo et al., 2005; Sánchez-Nieto et al., 2011).

This study did not analyse sugar transporter proteins during early or late germination times. Sucrose transporters from monocots are mainly expressed in mesophyll and phloem of source leaves, stems, developing seeds, and seedlings (Braun and Slewinski, 2009; Sun et al., 2010). ZmSUT1, the only SUT described in maize, has been characterized in seedlings and expressed in Xenopus oocytes, wherein mediates the bidirectional transport of sucrose (Carpaneto et al., 2005). The constitutive presence of this transporter in the germinating maize embryo implies that this protein is required for carbohydrate transport at this developmental stage.

As to the monosaccharide transporter proteins, Arabidopsis has at least 53 members grouped in seven subfamilies, of which two subfamilies codify hexose transporters, monosaccharide transporters, and sugar transporter proteins. Sugar transporter proteins have higher affinity for hexoses and pentoses with K _m values ranging from 10 to 100 μM (Büttner, 2007). Most of the hexose transporters are highly expressed in sink organs such as flowers and fruits, where they are thought to facilitate the acquisition of sugars unloaded into the apoplast (Hayes et al., 2007 and references therein) and in developing seeds (Weber et al., 1997; Raymond et al., 2003).

Sucrose transporter localization

In order to investigate whether membrane transporters were indeed allocated in regions where the sucrose was mobilized from, this study explored the physical localization of the sucrose transporter ZmSUT1. This was carried out in tissue slices from 24-h-imbibed embryos (Fig. 4). It was clearly observed that this transporter was present in the epidermis of scutellum cells (Fig. 4A) and associated with the plasma membrane in parenchymal cells (Fig. 4B, C) and, importantly, in the vascular tissue (Fig. 4D, E). By contrast, it was scarcely present in either the plumule of the embryo axis (Fig. 4F, G) or the epidermis and cortex of the root maturation zone (Fig. 4H). Localization was specific, because the stain was even lower in the control lacking the specific antibody (Supplementary Fig. S3). This differential ZmSUT1 localization suggested that the transporter was predominantly expressed in cells that are involved in the flow of sucrose from the scutellum symplast to the apoplastic spaces of the embryo axis and support the interpretation of the mobilization of sucrose (Table 1). In addition, these results are in agreement with the proposed physiological function of ZmSUT1 as a phloem loader (Braun and Slewinski, 2009), a concept derived from studies with knockout mutant plants that display low sucrose exportation, sucrose accumulation at the leaves, chlorosis, reduced growth, and impaired development (Slewinski et al., 2009).

Fig. 4.

Immunolocalization of the sucrose transporter in maize embryos. Embryos were imbibed for 24 h, fixed, infiltrated in sucrose, and frozen. Then, tissues were sliced into 8-μm sections and slices were processed for immunolocalization with the sucrose transporter antibody and viewed in a Nomarski differential interference contrast microscope. Red-brown coloration revealed a positive reaction with the sucrose transporter antibody. Arrows show the zones with the positive antibody reaction. Immunolocalization of sucrose transporter was strong in: (A) scutellum epidermis, (B, C) scutellum parenchyma cells, (D) scutellum vascular elements, and (E) vascular tracheidal elements at terminal development. Faint immunolabel is shown in (F) parenchymal cell of the plumule, (G) top view of the plumule, and (H) epidermal and parenchymal cells of root cortex. Immunodetection was performed in specimens from three independent experiments. Only representative views are shown. e, Epidermal cells; en, endosperm; p, parenchymal cells; t, tracheidal elements; v, vascular tissue. Bars, 33 μm (A–C, E–H) and 160 μm (D).

SUT and hexose transporters have been identified by using antibodies or determining their activities during the embryo development in Zea mays, Oryza sativa, Vicia faba, Pisum sativum, and Ricinus communis (Griffith et al., 1987; Weber et al., 1997; Aldape et al., 2003; Lim et al., 2006; Scofield et al., 2007b; Zhou et al., 2009). During the post-germination phase, the sucrose transporter has also been identified in the scutellum, aleurone, and companion cells of cereal seeds (Bick et al., 1998; Matsukura et al., 2000; Aoki et al., 2006; Scofield et al., 2007a). In such post-germination times, after radicle protrusion, it has been proposed that sucrose and glucose plus maltose, products of lipid metabolism in the aleurone layer and starch metabolism in the endosperm tissue, respectively, are taken up by the scutellum via a sucrose transporter and most likely an hexose transporter to synthesize the sucrose that will eventually go into the growing embryo (Aoki et al., 2006).

Medium acidification

As the electrochemical H⁺ gradient produced by the plasma membrane H⁺-ATPase drives secondary transport and apoplast acidification (Gaxiola et al., 2007), this study addressed the question whether this enzyme was required to promote the transport of the carbohydrates in the scutellum and the embryo axis and to produce the medium acidification required for radicle extension. Therefore, maize embryos were imbibed for different times and then transferred to a medium where changes in H⁺ concentration could be determined. Dry maize embryos initially produced an alkalinization of the medium, probably due to the release of some organic-base solutes (Fig. 5A). This alkalinization was progressively overcome with time (12, 18, and 24 h), when significant medium acidification arose.

Fig. 5.

Medium acidification and H⁺-pumping and ATP hydrolysis mediated by the plasma membrane H⁺-ATPase during embryo imbibition. (A) Medium acidification by whole maize embryos imbibed at the indicated times. (B) Plasma membrane H⁺-ATPase activity. Plasma membrane vesicles were purified from maize embryos imbibed at the indicated times and vanadate- or erythrosin B-sensitive ATP hydrolysis was measured as Pi release. (C) H⁺-ATPase H⁺-pumping activity. Plasma membrane vesicles were purified from maize embryos imbibed at the indicated times and then changes in external pH were determined by following the quenching of the fluorescence. Values are representative of at least four independent experiments.

ATPase and H⁺ pumping activities

To assess whether the plasma membrane H⁺-ATPase was associated with the observed medium acidification, the activity of this enzyme was determined, both as ATP hydrolysis and as proton-pumping activity, in PMV isolated from maize embryos imbibed at different times. ATP hydrolysis was evaluated as vanadate- or erythrosine B-sensitive activities, as these are two typical inhibitors of the P-ATPases (Ball et al., 1987) (Fig. 5B). Both inhibitors, independently added, decreased the total ATP hydrolysis by the same magnitude at all imbibition times tested (Fig. 5B), suggesting that the determined ATPase activity corresponded to the plasma membrane H⁺-ATPase. This activity was higher in the PMV obtained from the embryos imbibed for 18 and 24 h, as compared with that obtained from embryos imbibed for 6 and 12 h. A strong correlation was noted between the high ATP hydrolysis activity and high medium acidification at 18 and 24 h (Fig. 5A, B). Within this time-window, the phase II and phase III transition of water uptake takes place (16 h) (Fig. 1B), i.e. the time at which elongation of axial organs, such as the plumule and mainly the radicle, occurs (Fig. 1C).

To further prove H⁺-ATPase involvement in the acidification of the medium, the actual H⁺ pumping capacity of the PMV purified from imbibed embryos at the period between 6 and 24 h was estimated. The rate and extent of acidification driven by H⁺-ATPase were higher at 12, 18, and 24 h as compared to 6 h imbibition (Fig. 5C). The corresponding absolute ΔpH values also increased with imbibition time (Supplementary Table S1). Unfortunately, a quantitative comparison of the ΔpH values with analogous measurements by acridine orange absorbance changes (Galtier et al., 1988) or quenching of quinacrine fluorescence (De Michelis and Spanswick, 1986; Palmgren and Sommarin, 1989; Cowan et al., 1993; Grouzis et al., 1997) was not feasible, since no estimates of these parameters in terms of H⁺ concentrations or ΔpH values were provided. On the contrary, the values of ΔpH derived from the quenching of ACMA fluorescence in 6-day-old corn root seedlings (St. Marty-Fleurence et al., 1988) were 100-fold higher than those in the present study. However, it should be considered that growth is much more active in coleoptiles than in corn root seedlings and also that coleoptile membranes are fully functional. Moreover, the technical procedure worked well in the present study, as judged by the signal calibration of liposomes, and allowed the measurement of a ΔpH value of 1.0 unit (Supplementary Fig. S4). Therefore it is possible that the low ΔpH values shown by the PMV were due to the natural leakiness of these membrane vesicles, whose molecular architecture is in a reconstruction process because of the recent tissue rehydration at these early times of imbibition (Powell and Matthews, 1978; Sánchez-Nieto et al., 1997, 1998).

H⁺-ATPase content at the different times of imbibition remained unchanged (Fig. 3C, Supplementary Fig. S2) and, contrary to the observed levels of the sugar transporters, significant H⁺-ATPase content was present at 0 h (Fig. 3C). In this regard, it has been previously shown that the enzyme is already present at 0 h of imbibition with an activity differentially distributed throughout the embryo regions and that it is gradually replaced by different enzyme forms during the first 5 h of maize imbibition (Sánchez-Nieto et al., 1998; Enríquez-Arredondo et al., 2005; Sánchez-Nieto et al., 2011). Therefore, the increase in H⁺-ATPase activity at 18 h of imbibition was probably related to either the expression of a different isozyme, such as the MHA1 and MHA2 isoforms described in maize coleoptiles (Frías et al., 1996), or to post-translational regulatory mechanisms (Sondergaard et al., 2004). This is in consonance with the view that germination occurs with a set of enzymes that are already present in the seed (Srivastava, 2002).

Plasma membrane H⁺-ATPase localization

To determine the H⁺-ATPase distribution in the maize embryo, its immunodetection was carried out in slices from embryos imbibed for 18 h, the same time at which radicle extension occurs (Fig. 1C). H⁺-ATPase was found in several embryo regions – scutellum, radicle, mesocotyl, and epicotyl – all of which are involved in transmembrane transport and/or cell elongation (Fig. 6). The root cap showed immunostaining that increased its intensity from the columella towards the peripheral regions of the root cap, as well as the lateral regions, which are characterized by a high mucilage secretory activity (Fig. 6A). An important staining level was observed in the plasmalemma of the root cell epidermis (Fig. 6A, B) as well as in the parenchymal cortex cells (Fig. 6C) of the elongation and maturation zones of the root. Staining was totally abolished by suppression of the first antibody (Fig. 6D). These results showed that H⁺-ATPase was present in regions involved in radicle growth, which is consistent with the location of the activity of this enzyme in specific regions of the developing embryo to promote scutellum elongation in wheat (Rober-Kleber et al., 2003).

Fig. 6.

Immunolocalization of plasma membrane H⁺-ATPase in maize embryos. Embryos were imbibed for 24 h, fixed infiltrated in sucrose, and frozen. Then, tissues were sliced into 8-μm sections, incubated with the plasma membrane H⁺-ATPase antibody and viewed in a Nomarski differential interference contrast microscope. Red-brown coloration revealed a positive reaction with the H⁺-ATPase antibody. Arrows show the positive antibody reaction. (A) Root and cap root. (B) Root, transversal plane showing the epidermis and cortex cells. (C) Root cortex. (D) Control section (same region than in C, but in the absence of the H⁺-ATPase antibody). Immunodetection was performed in specimens from three independent experiments. Only representative views are shown. c, Cortex; cr, cap root; ep, epidermis; r, root. Bars, 500 μm (A), 50 μm (B–D).

Conclusions

Maize embryo germination has been characterized in terms of the most typical functional parameters. This well-characterized system was then used to analyse the role of the endogenous carbohydrates during the period that brings about radicle protrusion. Thereafter, protein detection and cell localization of the sucrose transporter, the sucrose content, sucrose and glucose uptake, and the features concerning the plasma membrane H⁺ pump and the hexose transporter presence were assessed. From these results, the following sequence of events emerged: the endogenous sucrose at the scutellum cells is discharged into the symplast, and then moves through the vascular tissue to the embryo axis cell apoplasts. There, sucrose is converted into hexoses by the cell-wall invertases. Then, glucose and fructose are translocated into the embryo axis cells by the hexose transporter for nourishment and to drive radicle protrusion. The time-dependent expression of the plasma membrane H⁺-ATPase activity correlates with the requirement of the generation of an electrochemical H⁺ gradient and acidification to support radicle elongation, which is in turn associated to the mobilization of sugars from the scutellum to the embryo axis by specific transporters.

Supplementary material

Supplementary data are available at JXB online.

Supplementary Materials and Methods : Detailed description of the indicated sections in Materials and Methods

Supplementary Table S1 : Equivalence between the quenching of ACMA fluorescence and the ΔpH formed in the plasma membrane vesicles obtained from embryos imbibed at different times

Supplementary Fig. S1 : Characterization of the plasma membrane sucrose and hexose transporter antibodies

Supplementary Fig. S2 : Levels of immunodetected sucrose and hexose transporters and plasma membrane H⁺-ATPase during embryo germination

Supplementary Fig. S3 : Negative controls of the sucrose transporter inmunolocalization

Supplementary Fig. S4 : Determination of medium acidification by quenching of ACMA fluorescence