Vicia faba cv. Witkiem plants (Nunhems Zaden BV, Haelen, The Netherlands) were grown in pots in a greenhouse at temperatures varying between 20°C and 30°C at 60–70% humidity and a 14/10-h light/dark period. Supplementary lamp light (model SONT Agro 400 W; Phillips Eindhoven, The Netherlands) resulted in an irradiance level of 200–250 μmol m⁻² s⁻¹ at the plant apex. Test plants were taken 3 weeks after germination (cf. Hafke et al., 2005).

Under the usually prevailing source-limiting conditions, the dynamic sucrose exchange along the transport phloem is controlled by pmf-driven transporters located at the plasma-membranes of SE/CCs and PPCs. Earlier studies pointed out that competitiveness between SEs and PPCs is determined by the electric component of the pmf (Hafke et al., 2005). Hence, membrane potentials of SE/CCs and PPCs were determined along the axis of intact Vicia faba plants after impalement by microelectrodes under microscopic surveillance as illustrated previously (Hafke et al., 2005).

To standardize the electrode positions along plants of various lengths, the plant length index (PLI) was introduced (Figure 1A). The position on the main vein of the youngest mature leaf was set to be PLI 1, a position halfway along the stem was defined as PLI 0.5 and the transition from the heterotrophic to the autotrophic region just above ground level corresponds to PLI 0. The resting potentials of SE/CCs and PPCs were recorded at the respective plant axis positions and the membrane potential ratios V_{m SE/CC}/V_{m PPC} calculated (Figure 1B).

The average membrane potentials of SE/CCs (Figure 1B) varied between −127 mV at PLI 1 and −112 mV at PLI 0 and those of the PPCs remain around −100 mV along the entire stem stretch. The distinct and consistent difference between the membrane potentials of SE/CCs and PPCs at various PPIs indicated a symplasmic disjunction as found before in transport phloem (Oparka et al., 1994; van Bel and van Rijen, 1994; Kempers et al., 1998; Hafke et al., 2005). Since the membrane potentials of the SE/CCs declined more rapidly along the plant axis than those of the PPCs (see ratio, Figure 1B), the relative competitiveness of the PPCs may increase towards the stem base.

Membrane potentials were also recorded in SEs and PPCs embedded in free-lying vascular root tissue. There, the SE membrane potentials are drastically reduced to −55 ± 2.4 mV (n = 4) in comparison with SEs in the stem. No stable values for PPCs could be obtained so that no conclusions could be drawn regarding the relative competitiveness between SEs and PPCs in roots.

As discussed before (Hafke et al., 2005), kinetics of sucrose uptake by SE/CCs and PPCs is a key element in the competitiveness between SE/CCs and PPCs and, hence, for photoassimilate partitioning. However, the cell-specific uptake kinetics has not been determined conclusively to date. Studies of ¹⁴C-sucrose uptake by phloem strips do not discriminate between the kinetics of SE/CCs and PPCs. Furthermore, uptake studies using SE- and PPC-protoplasts would require an immense amount of protoplasts which is virtually impossible given the painstaking isolation and identification procedures. Therefore, we measured membrane depolarizations of single cells induced by various apoplasmic sucrose concentrations under physiological conditions (e.g., Lichtner and Spanswick, 1981). Natural apoplasmic sucrose concentrations are in the range between 1.0 and 60.0 mM (cf. Patrick and Turvey, 1981; Minchin and Thorpe, 1984; Voitsekhovskaja et al., 2000; Kang et al., 2007).

As reported before (Hafke et al., 2005), microelectrodes were impaled into SEs (Figures 1C,D) or PPCs (Figure 1G). Following stabilization of the membrane potential, various sucrose concentrations were supplied to SE/CCs by bath perfusion (for SEs) or local micropipette-mediated solute administration (for PPCs). Electrical recordings showed graded effects of various sucrose concentrations on the membrane potentials of SEs (Figure 1C) and PPCs (Figure 1G). As a general pattern, a fast sucrose-induced depolarization brought about by H⁺ co-transport (e.g., Carpaneto et al., 2005) was followed by a gradual repolarization. Replacement of the sucrose solutions by an iso-osmotic mannitol solution accelerated the repolarization to the resting potential (Figure 1C).

At a decreased external pH (3.5 instead of 5.7), application of the same sucrose concentration (10 mM) resulted in a 3-fold increase in the magnitude of depolarization (Figure 1D) which is in agreement with the role of protons in pmf-driven H⁺-sucrose symport (e.g., Delrot and Bonnemain, 1981; Reinhold and Kaplan, 1984).

The relationship between sucrose concentration and SE or PPC depolarization can be described by single Michaelis–Menten kinetics (MM) or a biphasic Michaelis–Menten kinetics (2 MM) (Figures 1E,H). Regression diagnostics (sum of squared residuals SSRs, R²) from non-linear least-square data fitting (NLSF) revealed a slightly better fit to 2 MM as indicated by smaller SSRs and a higher R² as compared to MM (Table 1). Furthermore Eadie–Hofstee (EH) transformations revealed two linear components (Figures 1F,I) which are indicative for two separate, simultaneously operating uptake systems (e.g., Reinhold and Kaplan, 1984). The K_m values of either uptake system can be calculated from the linear regression at low and high sucrose concentrations.Unmistakable biphasic uptake kinetics are visible for the SE data at PLI 0.5 (Figure 1F, Table 1), but those at PLI 1 and PLI 0, data can be interpreted to be due to either MM or 2 MM (Figure 1F, Table 1).

Diagnostic tools like SSR and R² values alone do not allow predictions on the adequacy of a kinetic model (MM or 2 MM). Akaikes Information Criterion (AIC, Motulsky and Christopoulos, 2003; Burnham and Anderson, 2004) was applied to test for the best model (Table 2). MM is more likely to be correct then 2 MM which is indicated by smaller AIC_c values for the MM model (Table 2). The small difference in Δ_I of <2 at PLI 0.5 (1.82) indicates that 2 MM has a substantial support (evidence) whereas models having Δ_I > 10 (in case of 2 MM at PLI1 and PLI0) have essentially no support (Burnham and Anderson, 2004). Fitting the data to MM would render a shift in K_m values for SEs along the plant axis (Table 1). K_m values obtained by NLSF increased from 1.86 mM (EH: 1.73 mM) at PLI1 to 6.32 mM (EH: 9 mM) at PLI 0 (Table 1) suggesting a decreased sucrose retrieval by SEs along the plant axis.

For PPCs, a K_m value of 52 ± 26.5 mM showing a high standard error was calculated (Table 1) when MM was adopted as the mode of uptake. Using 2 MM, Eadie-Hofstee calculations produced K_m1 and K_m2 values of 10 and 70 mM, respectively, while NLSF revealed a K_m1 value of 1.45 mM and infinitesimal high K_m2 (Table 1). Irrespective of the mode(s) of uptake, it suggests that the affinity for sucrose uptake is much lower in PPCs than in SEs.

In analogy to studies of the phloem-localized sucrose carrier ZmSUT1 expressed in Xenopus oocytes (Carpaneto et al., 2005), patch-clamp technique was applied to SE protoplasts in the whole-cell configuration (cf. Hafke et al., 2007) for a direct detection of the sucrose/H⁺ transporter activity. SE protoplasts (Figure 2A) were bathed in a sucrose-free medium (pH 5.5; Figure 2B) and clamped to a holding voltage of −106 mV. In response to external supply of 100 mM sucrose (pH 5.5) by local bath perfusion, an increase in inward current (positive current into the cell, cf. Bertl et al., 1992) was recorded. Normalized currents were in the range of −0.1 to −0.3 pA/pF (−0.2 ± 0.07 pA/pF, n = 4). This time-dependent increase in negative currents (downward deflections, Figures 2C–E) represent proton currents generated by a H⁺/sucrose symporter mediating sucrose transport into SEs as reported for ZmSUT1 expressed in Xenopus oocytes (Carpaneto et al., 2005).

Kinetic parameters for sucrose uptake are crucial for the competition between SE/CCs and PPCs in transport phloem and the distribution of photoassimilates over axial and terminal sinks (van Bel, 1996; Hafke et al., 2005). Other than in previous approaches, K_m values for sucrose uptake were calculated here by measuring sucrose-induced membrane depolarizations of SEs and PPCs (Figure 1) in analogy to an approach using cut aphid stylets in combination with electrophysiology (Wright and Fisher, 1981). As aphid stylets are solely inserted into SEs, the latter technique is not applicable for PPC recordings.

The sucrose-induced depolarizations are consistent with H⁺-sucrose co-transport with a 1:1 stoichiometry (e.g., Lichtner and Spanswick, 1981; Wright and Fisher, 1981; Carpaneto et al., 2005). The magnitude of depolarization varies with external sucrose concentrations in keeping with Michaelis–Menten uptake kinetics (Figures 1E,H). The K_m values calculated for uptake by sieve elements (Table 1) match well with those obtained by uptake studies in oocytes and yeast (Figure 3) which can be regarded as proof for the adequacy of the present electrophysiological approach. The K_m values (Table 1) are higher than those observed for sucrose uptake by isolated Commelina minor veins (0.5 mM), but determination of the parameters of active uptake may have been confounded by an appreciable diffusional uptake component in this study (van Bel and Koops, 1985).

Plotting the relationship between sucrose concentration and membrane depolarization can be described by either single (MM) or biphasic Michaelis–Menten kinetics (2 MM). As for SEs, Eadie-Hofstee transformations revealed some evidence in favor of two uptake systems (Figure 1F, Table 1), but the differences in significance between MM or 2 MM were marginal. To distinguish between MM and 2 MM, Akaike's Information Criterion (AIC)-based model selection was invoked for further analysis (Table 2). As a result, MM is favored for SEs at PLI 1 and PLI 0 (Table 2), whereas AIC supported also 2 MM for SEs at PLI 0.5 (see also Figure 1F) by a difference in AIC_c values between MM and 2 MM of Δ_i < 2 (e.g., Burnham and Anderson, 2004).

The inconclusive assessment of the uptake mode(s) which may also be due to the limited data sets, leaves us with two possible interpretations:

Only one single active uptake system is involved, but MM kinetics is distorted by artifacts induced by the experimental approach. Uptake may be influenced by unstirred layers in vicinity of the plasma membrane as described for intestinal studies (Thomson, 1977; Thomson and Dietschy, 1977; Gardner and Atkinson, 1982). The absorption of solutes in anatomical complex tissues leads to concentrations in the close vicinity of the cell membrane being lower than that in the bulk fluid leading to a deformation of the Michaelis–Menten kinetic or the Eadie-Hofstee transformation and a serious shift in K_m of the sucrose uptake system to higher values (Winne, 1977; Thomson, 1977; Thomson and Dietschy, 1977; Gardner and Atkinson, 1982). Diffusion through the cell-wall microfibrils may be limited by physical forces in the cell-wall micro-environment.

A further pitfall causing dual kinetics may be symplasmic coupling in intact tissues: biphasic kinetics could be ascribed to differential contributions of electrically coupled cells. Therefore, symplasmic isolation is an absolute prerequisite for a correct interpretation of the data. Fluorochrome studies demonstrated that SE/CCs in transport phloem are strictly disjunct from PPCs under the present conditions (van Bel and van Rijen, 1994; Patrick and Offler, 1996; Knoblauch and van Bel, 1998; Hafke et al., 2005), when the few plasmodesmata in the SE/CC-PPC interface (Kempers et al., 1998) are closed. By contrast, SEs and CCs are strongly coupled in situ via numerous PPUs (Kempers et al., 1998). However, electrode impalement likely confers occlusion by PPUs (Knoblauch and van Bel, 1998) so that the biphasic depolarization patterns are to be merely ascribed to SEs. For PPCs, it remains uncertain to which degree plasmodesmata towards adjacent cells are closed after microelectrode insertion.

The uptake mode(s) has(have) a strong impact on sucrose processing along the phloem pathway. The existence of one sucrose uptake system in SEs of transport phloem would disclose a shift in the K_m values of SEs from 1.8 mM to approx. 6 to 7 mM downwards along the plant axis (Table 1, NLSF, EH for MM). In parallel with the increase in the affinity constant, membrane potentials in SEs decline along the plant axis (Figure 1A), leading to reduced pmf-driven sucrose uptake (e.g., Hafke et al., 2005). A comparable voltage-dependence of K_m values was already characterized for the sucrose carrier ZmSUT1 expressed in oocytes (Carpaneto et al., 2005). Carpaneto et al. (2005) showed, that that K_m values for ZmSUT1 increased at more positive membrane voltages. For PPCs showing much higher K_mvalues for sucrose uptake (>10 mM; Table 1), more detailed data analysis on kinetic properties of sucrose uptake is necessary. Recently, two efflux carriers mediating a key step in phloem loading have been characterized in Arabidopsis: AtSWEET11 and AtSWEET12. These two sucrose transporters are highly expressed in leaves and are both localized in the plasma membrane of phloem parenchyma cells (Chen et al., 2012). The atsweet11/atsweet12 double insertional mutant plants are defective in phloem loading and display a phenotype similar to AtSUC2 knock-out mutants with regard to sugar and starch accumulation (Gottwald et al., 2000). Both AtSWEET11 and AtSWEET12 seem to be different from the other proteins belonging to the SWEET family because of their low affinity to sucrose, with the K_m values for an influx ≈70 and efflux >10 mM (AtSWEET12). Because of the pH-independent transport, it was suggested that the sucrose translocation may rely on a uniport system (Chen et al., 2012).

It should be noted, that, pmf-driven uptake rates may be constant, since membrane potentials of PPCs did not change along the plant axis (Figure 1B). Both membrane potential gradients and K_m gradients infer that the competitiveness of sieve elements declines downwards along the axis.

Carbohydrate partitioning and management strongly depend on the release/retrieval of photoassimilates along the phloem path under source-limiting conditions (Patrick and Offler, 1996; van Bel, 2003a,b; Hafke et al., 2005). The rate of (temporary) diversion of photoassimilates to axial sinks is determined by a competition between SE/CCs and PPCs (Hafke et al., 2005). According to the present data, the relative competitiveness of PPCs tends to increase near the stem basis (ratio, Figure 1B). According to the K_m-values assuming one sucrose uptake system along the axis (Tables 1, 2), the competitiveness of SE/CCs is higher at low apoplasmic sucrose concentrations than at high concentrations which can be handled more easily by PPCs (having low-affinity uptake system). Under sink-limiting conditions, the amounts of photoassimilates available for the axial sinks are extremely high. Then, membrane uptake systems will be of minor importance since most of the photoassimilates move through open plasmodesmata towards the PPCs (Patrick and Offler, 1996). Under these circumstances, the low-affinity systems of PPCs may be quite useful for retrieval of massive amounts of sucrose leaking from the axial sink cells.

In previous studies, ZmSUT1 was heterologously expressed in oocytes and characterized functionally in detail using a patch-clamp approach (Carpaneto et al., 2005, 2010). The sucrose-coupled proton currents were reversible depending on the direction of the sucrose and pH gradients and the apparent affinity constant K_m of ZmSUT1 exhibited a pronounced voltage and pH-dependence (Carpaneto et al., 2005). The turnover rate of ZmSUT1 at a physiological membrane voltage of −120 mV is about 500 s⁻¹ (Carpaneto et al., 2010). Based on this value, a transporter density of 10⁴/μm² was calculated for oocytes expressing ZmSUT1 (Carpaneto et al., 2010), which exceeds by far the transporter densities to be expected in plasma membranes under natural conditions.

Using a similar patch-clamp approach (cf. Carpaneto et al., 2005), a sucrose/H⁺ symport activity was detected in the plasma membrane of SE protoplasts (Hafke et al., 2007) as indicated by an increase in inwardly directed currents (visible as downward deflections relative to the baseline) in response to sucrose supply. Neglecting possible rundown effects previously described for the H⁺/sucrose symporter ZmSUT1 (Carpaneto et al., 2005), negative currents with current densities I/C_m around −0.2 pA/pF (Figure 2) were recorded in the presence of 100 mM sucrose, a membrane voltage of −106 mV and a gradient of 2 pH units. The observed current densities lie in the order of magnitude of sucrose-induced H⁺ currents (Schulz et al., 2011; SUC4 transporters) or myo-inositol driven H⁺ currents (Schneider et al., 2008) in vacuoles of Arabidopsis thaliana. To date, sucrose carrier activity in native membrane systems were measured solely in isolated vacuoles by the patch-clamp technique (Schulz et al., 2011). Given the detectable H⁺/sucrose symporter activity in the plasma membrane of SEs (Figure 2), SE protoplasts might become a suitable tool to characterize sucrose transporters in their natural membrane environment and hence complement the data set obtained with transporters expressed in heterologous systems like oocytes (Carpaneto et al., 2005, 2010).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.