Journal of Experimental Botany, Vol. 51, No. 347, pp. 1047-1055,
June 2000
© 2000 Oxford University Press
Effluxes of solutes from developing seed coats of Phaseolus vulgaris L. and Vicia faba L.: locating the effect of turgor in a coupled chemiosmotic system
1 Department of Biophysics, School of Physics, University of NSW, Kensington, NSW 2052, Australia
2 Department of Biological Sciences, University of Newcastle, Newcastle, NSW 2308, Australia
Received 6 June 1999; Accepted 21 January 2000
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
|---|
Cells lining the developing seed coats of legumes efflux photosynthates (mostly sucrose) and salts (mostly of potassium) into the apoplast for uptake by the developing embryo. These effluxes increase transiently in response to an increase in turgor in the effluxing cells. Detached coats of developing seed of Phaseolus vulgaris and Vicia faba were used to study the effects of turgor on the rates of efflux, on the membrane potential difference and on the membrane pH difference, using a number of inhibitors and agents which might affect signal cascades involving cytoplasmic calcium concentration. Effluxes were measured by measuring the concentrations of solutes of interest in solution samples placed in halves of detached seed coats, the paired halves serving as control and treated sample where appropriate. It is shown that a number of substances affect sucrose and potassium effluxes differently, and that hypo-osmotic shock depolarizes the efflux cells and acidifies the cytoplasm (in P. vulgaris). It is concluded that sucrose and potassium effluxes, although both are increased by an increase in turgor, are affected by different signal pathways. Further, it is also concluded that the signal that increases the rates of both sucrose efflux (via sucrose–proton antiport) and proton pump acts directly on the antiporter rather than on the pump. There are interesting parallels and contrasts between these processes and those in plants such as the charophyte Lamprothamnium after hypo-osmotic shock.
Key words: Potassium, sucrose–proton antiport, efflux, seed coat, turgor.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
|---|
In developing seeds of the legumes Phaseolus vulgaris L. and Vicia faba L. there are solute effluxes from the seed coats into the apoplast surrounding the cotyledons. The solutes are photosynthates (largely sucrose in both P. vulgaris, Patrick, 1984
, and V. faba, Offler et al., 1989) and salts (largely of K+, Patrick, 1984; Walker et al., 1995). These effluxes are increased when the turgor in the unloading cells of the seed coats is increased above its normal value of about 2 kPa (Patrick, 1994a; Zhang et al., 1996). This increase in efflux appears to be the mechanism of downward short-term turgor regulation in the unloading cells (Zhang et al., 1996). A different mechanism is thought to regulate turgor upward (Thorpe et al., 1993). Comparison between attached and detached seed coats led us to the conclusion that upward regulation may result from increased solute import through the phloem (Zhang et al., 1996). It is consistent with this that attached seed coats can regulate cellular turgor in both downward and upward directions, while detached seed coats (as used here) can regulate downwards but cannot regulate upwards (Zhang et al., 1996). The existence of such upward and downward turgor regulation in attached seed coats is an important prediction of the turgor-homeostat model for control of efflux (Patrick, 1994b).
In the unloading cells of seeds of bean (P. vulgaris and V. faba) it was shown that there is a chemiosmotic system in which the H+-pump maintains an inward proton-motive force (PMF) that drives sucrose antiport and probably also passive chloride efflux (Walker et al., 1995), although pea (Pisum sativum) may well be different (De Jong et al., 1996). Walker et al. showed that the pump and antiport interact kinetically through the plasma membrane pH difference (
pH), rather than through the total PMF or through the membrane electric potential difference (PD) (Walker et al., 1995). It is argued that K+ efflux could be passive through plasma membrane channels. Following this, time-dependent K+ outward rectifiers (KOR) and time-independent rectifiers (IOR) in V. faba (W-H Zhang et al., 1997) and in P. vulgaris (Zhang et al., unpublished results) have been characterized and it was and concluded that they did not show sufficient conductivity to explain the normal (low turgor) unloading flux of K+. It has previously been shown in both P. vulgaris (Patrick, 1984) and V. faba (Patrick, 1994a) that elevated turgor increases the efflux of K+: here the likely role of the IOR and KOR channels has been re-examined.
In an earlier paper (Walker et al., 1995) a new method of analysis was introduced in which numbers of biochemical agents were applied with the intention of stimulating or inhibiting one or other coupled process, and then looked for correlations in the data. Reliance was placed on the biochemical agents to produce a spread of data values, but no reliance on any one agent to act as expected. This proved a fruitful method, and a similar approach is used in the present paper.
This work sets out to discover where elevated turgor acts in the coupled chemiosmotic system, continuing the chemiosmotic analysis of potassium and sucrose effluxes. In the present paper the effects of elevated turgor on the relationship between the H+-pump and the sucrose antiporter are examined. To this end a range of inhibitors, channel blockers and ionophores were used for studying turgor action on K+, H+ and sucrose efflux, and changes in membrane PD and cytoplasmic pH were measured under imposed turgor changes. Many experiments were carried out on both P. vulgaris and V. faba, while for various practical reasons some were or could be carried out on one or the other only.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
|---|
Plant material
Plants of Vicia faba L. (cv. Coles' Dwarf Prolific) and Phaseolus vulgaris L. (cv. Redland Pioneer) were raised under glasshouse conditions with partial temperature control (20–26 °C by day and 18–24 °C by night). Plants were grown singly in 3.0 l of potting mixture (for details, see Fieuw and Patrick, 1993
). Mineral nutrients were supplied in half-strength Hoagland's No. 1 solution at the rate of 100 ml per pot per week. To get optimum pod set and subsequent seed growth, plants were pruned as follows. For V. faba, shoot axes were decapitated above the fifth flowering node. After pod set, pod numbers were reduced to one per node. For P. vulgaris, plants were pruned to leave the primary and second trifoliate leaves supporting a single pod arising from the axis of trifoliate leaf 3. For both species, the pruning regime ensured that seed growth was sink-limited.
Two to three weeks after anthesis, the V. faba plants were transferred to growth chambers set for 19 °C by day and 15 °C by night. The PAR incident on the upper leaves was stepped as follows: 0–2 h at 60; 2–4 h at 200; 4–10 h at 400 µmol m-2 s-1 and thereafter the reverse sequence to give a 14 h photoperiod. The P. vulgaris plants completed their entire development under the glasshouse conditions described above. However, 18 h prior to experimentation, the P. vulgaris plants were transferred to controlled environmental conditions of a temperature of 21 °C, a photoperiod of 14 h and an incident PAR of 150 µmol m-2 s-1. Developing seed were used for experimentation when cotyledon expansion growth was approaching completion but dry weight gains were rapid and linear. The developmental stage of the seed used was early-to-mid Phase II for V. faba (Briarty et al., 1969) and developmental Stage IV for P. vulgaris (Walbot et al., 1972).
Procedures and solutions
Solutions and procedures were identical to those given previously (Walker et al., 1995) except for necessary modifications as specified. Unless specified otherwise, external osmotic concentration (EOC) was set by adding sorbitol at the following values: P. vulgaris, normal=300 mOsM; low=80 mOsM; V. faba, normal=420 mOsM; low=120 mOsM. The ‘normal’ values of EOC were close to the in vivo values measured in seed coat apoplastic fluid (Zhang et al., 1996; JW Patrick, unpublished results).
Metabolic agents were used at the following final concentrations: Gd3+, 5 mM; La3+, 10 mM; A23187, 100 µM; cyclopiazonic acid (CPA), 50 µM; N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7), 100 µM; chlorotetracycline (CTC), 500 µM; ethylene glycol-bis(ß-aminoethyl ether) N,N,N,N'-tetraacetic acid (EGTA), 0.1–0.25 mM, phlorizin (PZ), 5 mM. These concentrations were chosen after preliminary experiments to be the lowest that showed consistent effects. CPA and A23187 were dissolved in DMSO to a final concentration of 0.1%, and DMSO was added to the respective controls to the same final concentration. EGTA was dissolved in a small volume of 1 M NaOH, and the final pH was adjusted. CPA solutions were kept in the dark.
Efflux measurements
Efflux measurements were made as described earlier (Walker et al., 1995). Detached seed coats were halved along a line through the hilum, the cotyledons were removed, and each half seed coat was filled with experimental solution. The paired halves served as treated and control where this was appropriate. In the sucrose and H+ flux experiments (e.g. Fig. 1) the solutions were changed at 20 min intervals in order to build up a measurable solute concentration, and replicates consisted of four seed-coat halves. In 14C-labelled photosynthate experiments solutions were changed at 2 min intervals and efflux from each seed-coat half was measured. All fluxes are reported in terms of dry weight of tissue.
|
, •) and potassium (
, 
) effluxes from excised seed-coat halves, measured over successive 20 min periods. Control (open symbols), and after step-down in EOC (closed symbols). (a) P. vulgaris (—) and (b) V. faba (–··–··). Error bars represent SEM, n=4, each replicate pooled from four seed-coat halves.The technically more difficult measurement of sucrose efflux was chosen where it was considered that the absolute value was significant: i.e. for comparison with the H+ or K+ efflux (see Figs 1
, 2, 3). Where the aim was to measure degrees of inhibition, the simpler measurement of 14C-assimilate efflux was chosen, based on the finding that 80% of this efflux is sucrose (Patrick, 1984, for Phaseolus; Offler et al., 1989, for Vicia). Measurement of 14C-assimilate efflux carried the bonus that a separate value could be obtained for each seed-coat half, allowing paired comparisons between halves of the same coat.
|
|
) efflux from excised seed coat halves of P. vulgaris (—) and V. faba (–··–··) after a step-down in EOC. Efflux in the presence of PCMBS expressed as a percentage of control in its absence. Measurements made 20–30 min after step-down in EOC. Each individual seed coat provided a control half and a PCMBS-treated half, yielding one value of efflux in PCMBS as a percentage of control, at one value of EOC. Plotted values are global means of five means of groups of eight such values. The arrow indicates the starting EOC, the direction of change, and an approximate EOC step-down to give 500 kPa turgor step up. Error bars represent SEM; n=8.
PD measurements
Slices of seed coats of P. vulgaris were bathed in 0.5 mM KCl, 5 mM MES, 1 mM CaCl2 (pH 6.5) with initial sorbitol concentration of 300 mM. After the microelectrode insertion, once the PD became steady, the sorbitol concentration of the bathing solution was stepped to 200, 100 and 0 mM in sequence. The PD was monitored during these sequential changes of bathing solution and the values given are the approximately steady PDs achieved usually 10 min after each change of bathing solution, the experiment thus occupying about 1 h. Because microelectrode insertion was technically difficult, separate seed coats could not be used for each step in EOC, as in the other experiments reported here. In the absence of changes in EOC the PD remained constant for at least 1 h. The difficulty of obtaining similarly steady PDs with V. faba prevented useful results being got with that species.
Cytoplasmic pH measurements
Average cytoplasmic pH was measured as previously described (Walker et al., 1995), allowing 5,5'-dimethyl 2,4-oxazolidine dione (DMO) to equilibrate for the 60 min following the change in EOC.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
|---|
Sucrose, H+ and K+ fluxes
The effect of a step-down (from normal) in EOC on the effluxes of sucrose and K+ from detached seed coats is shown in Fig. 1
for both P. vulgaris and V. faba. The efflux values were measured over 20 min periods starting 10 min after the change in solution. By this time the initial inward water flow had ceased (cf. Patrick, 1984) and the initial rise in efflux was complete (not shown, as it was obscured by washout from the apoplast, Patrick, 1994a). In the controls (open symbols) the efflux of K+ appears steady in both species, and that of sucrose falls noticeably with time. This fall may be due to sucrose depletion in the freshly-detached seed coat. The greater falls in efflux under high EOC treatment (solid symbols), in all but K+ in V. faba, may be due to the increased efflux both reducing the turgor error signal (Patrick, 1994b) and also depleting the efflux substrate.
The fall in efflux shown in Fig. 1 is considerably slower than the recovery in cell turgor measured directly (Zhang et al., 1996). Since the direct measurements of cell turgor used much smaller changes in turgor (about 70 kPa) than the present flux measurements (of the order of 500 kPa), the data are not, however, directly comparable.
Other measurements reported here were made as early as practicable in the time-course, over the period from 10 to 30 min after the step-down unless otherwise stated.
Figure 2 shows, for both P. vulgaris and V. faba, the effect of the size of the step-down in EOC (from 400 mOsM) on the effluxes of sucrose and K+. For all steps down there is a progressive rise in each efflux. It is notable that sucrose and K+ effluxes respond very similarly to turgor changes. These observations are consistent with those on photosynthate efflux in both species by Patrick (1994a).
The effect of the inhibitor p-chloromercuribenzenesulphonate (pCMBS) at 5 mM on the efflux of 14C-labelled photosynthate at various EOC is seen in Fig. 3 for both P. vulgaris and V. faba. The points plotted are means of five experiments. The inhibition by PCMBS is constant (about 40%) down to 100 mOsM for P. vulgaris, and (about 50%) down to 50 mOsM for V. faba, but it falls off as EOC is further reduced. At still lower EOC the PCMBS-insensitive component becomes dominant in P. vulgaris, as also observed by Patrick et al. (Patrick et al., 1986). This is consistent with the large rise in sucrose efflux seen in Fig. 2a for P. vulgaris.
For other work on excised seed coats, concentrations of 80 and 120 mOsM were chosen for P. vulgaris and V. faba, respectively, to produce moderately elevated turgor; since at these concentrations (Fig. 2) a marked increase in efflux can be seen and the increased efflux (Fig. 3) is inhibited by PCMBS to about the same extent as the normal efflux.
The effects on photosynthate efflux were investigated of these steps down in external osmotic concentration and of the pump inhibitor erythrosine B (EB), the sugar transport inhibitor phlorizin (PZ) and the external sulphhydryl reagent PCMBS. The data (Table 1) show that a component of normal and turgor-increased photosynthate efflux is sensitive to PCMBS and also to PZ, a specific inhibitor of sucrose porters (Bush, 1993), and EB, an inhibitor of the H+ pump (Walker et al., 1995; Beffagna and Romani, 1988).
|
Table 2
shows that EB and PZ also affect net acid influx into seed coats of both species, the former increasing it and the latter reducing it both in controls and when the efflux is increased by a step-down in EOC.
|
Membrane PD
The effect of a step-down in EOC on the membrane PD of unloading cells of P. vulgaris, in a medium containing 0.5 mM added potassium is shown in Fig. 4. In this solution [K+] is calculated to be equivalent to 2 mM at the unloading cell membrane (cf. Walker et al., 1995), compared with about 1.5 mM for the other experiments reported here. A consistent further depolarization is seen to be produced by the step-down in EOC, the PD reaching -70 mV at about zero EOC. For a step-down to the EOC used as ‘low’ for P. vulgaris in the other experiments reported here (80 mOsM) the depolarization is estimated as 23 mV. The PD at normal turgor approaches -100 mV, and so is consistent with the value of -95 mV found previously under similar conditions (Walker et al., 1995) and close to the calculated value of the K+ equilibrium potential -103 mV (Fig. 4).
|
) of unloading cells of P. vulgaris (—) in a medium containing 0.5 mM added K+ (equivalent to 2 mM K+, see text). Measurements made 10 min after each solution change. Error bars represent SEM; n=8. The arrow indicates the starting EOC, the direction of change, and an approximate EOC step-down to give 500 kPa turgor step up.There are interesting features of the PD results:
- (i) that the PD falls slowly after a step-down in EOC (half-time about 5 min, data not shown)
- (ii) that the cells remain depolarized or become more depolarized during the period when the turgor is (at least partly) adjusted (cf. Zhang et al., 1996
)
- (iii) that the PD does not recover when the EOC is restored to normal (data not shown). Impaled cells not subjected to turgor increase hold a constant PD for 1 h, so the failure to repolarize is not seen as an artefact of injury.
- (ii) that the cells remain depolarized or become more depolarized during the period when the turgor is (at least partly) adjusted (cf. Zhang et al., 1996
Membrane pH
Table 3 shows the results of DMO distribution experiments to determine the mean effective cytoplasmic pH of the cells of seed coats of P. vulgaris and V. faba. The effect was investigated of a step-down in EOC, but for DMO equilibration needed to allow the relatively long period of 60 min. These results are thus averaged both over several different cell types (see below) and over a period in which turgor is being regulated and fluxes are returning to normal (e.g. Fig. 1). It was found that the step in EOC produced a drop in mean tissue cytoplasmic pH in P. vulgaris of 0.24 pH unit, but there was no detectable change in cytoplasmic pH in V. faba.
|
For P. vulgaris, it was assumed that efflux, turgor regulation and hence pH shift is confined to the unloading cells. These occupy 40% of the tissue volume (JW Patrick, unpublished results), and if their cytoplasm is also assumed to occupy 40% of the tissue cytoplasm volume, the actual shift is 0.45 unit, time-averaged over the 60 min. However, in V. faba the unloading cells are estimated to contain only 7% of the tissue volume (JW Patrick, unpublished results). If they also contain 7% of the tissue cytoplasm volume then a shift of 0.45 units in their cytoplasmic pH would give a mean tissue shift of only 0.05 unit, which is undetectable by this method.
Calcium-altering agents
Because Ca2+ plays a central role in signalling in many plant systems (Okazaki and Tazawa, 1990; MacRobbie, 1993; Cote, 1995), a number of agents reputed to disturb concentrations or fluxes or actions of Ca2+ were selected. These were: two members of the lanthanide series, Gd3+ and La3+ (Tester, 1990); the calcium ionophore A23187; the calcium ATPase inhibitor CPA (Thompson et al., 1994); the calcium chelator EGTA; the ‘calmodulin antagonist’ W-7 (Laver et al., 1997) and the Ca2+ binding agent CTC (Tsein, 1989).
Measurements were made for V. faba of the inhibition of the effluxes of K+ and of 14C-labelled photosynthate under normal and step-down conditions. Representative results are shown in Table 4. In examining the results the authors are looking for overall patterns and not relying on the reputed actions of these agents in other systems,. First, it is notable that Gd3+ and La3+ affect both photosynthate and K+ effluxes, contrasting with EGTA and A23187+CPA which affect only photosynthate efflux and with W-7 and CTC which affect only K+ efflux. A second contrast is that K+ efflux from normal and stepped-down seed coats is similarly affected by all the agents used, while photosynthate efflux from normal seed coats is more inhibited by Gd2+, La3+ and EGTA and less inhibited by A23187+CPA, than that from stepped-down coats. The only example of an effect on a turgor-stimulation, rather than on an efflux as such, is the inhibition of turgor-stimulation of photosynthate efflux by A23187+CPA.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
|---|
EOC and turgor
In this study no direct measurements of turgor were made, so the results have been reported in terms of changes in EOC intended to alter cell turgor. A change in EOC from a normal value of 300 to 80 mOsM would increase turgor by up to 490 kPa, and one from 420 to 120 mOsM would increase it by up to 670 kPa. It needs always to be borne in mind that steps down in EOC as used here give steps up in turgor.
Effects of turgor increases
It is a feature of the results in Fig. 2 that sucrose efflux and K+ efflux respond similarly to rises in turgor, including the larger rise at highest turgor seen in P. vulgaris though not always in V. faba. It is noteworthy that the actual increase in efflux of K+ is very similar to that of sucrose under most conditions (Figs 1, 2). The net H+ influx (Table 2) is much smaller than the effluxes of sucrose and K+ (Figs 1, 2). Allowing for the anions therefore that must accompany the K+, ion efflux accounts for about twice as much turgor regulation as sucrose efflux does. The very fact of net proton influx raises unanswered questions: is it balanced electrically by net K+ efflux? Is this exchange balanced chemically by K+/H+ exchange at the tonoplast? What determines the rate of these processes?
K+ efflux mechanism and membrane PD
The turgor step increases the efflux of K+ (Fig. 2) by the equivalent of about 90 nmol m-2 s-1 in P. vulgaris and 34 nmol m-2 s-1 in V. faba. The depolarization of 23 mV under these conditions (measured in P. vulgaris, Fig. 4) is in the appropriate direction for the increase in efflux to be the result, not the cause, of the PD change. However, the results on P. vulgaris protoplasts (W-H Zhang et al., unpublished results) suggest that a depolarization from -95 mV to -70 mV (Fig. 4) would produce an increase in K+ efflux of only about 10 nmol m-2 s-1, through KOR or IOR, in 1 mM external K+. This is only about 10% of the increase observed. There are no PD data for V. faba., but the results on its K channels, especially the IOR (Zhang et al., 1997), suggest that a depolarization of similar size to that in P. vulgaris, starting from somewhere near EK, might produce the additional K+ efflux through the IOR. This rectifier (channel) seems, however, unlikely to produce the normal efflux, which, like the fluxes in P. vulgaris, may well be by an electrically silent mechanism such as K+/H+-antiport (Felle, 1989; Cooper et al., 1991). The parallels in Table 4 between effects on normal and enhanced K+ efflux in V. faba suggest that, in this species, the mechanisms of normal and enhanced efflux may well be the same. It seems that in both species the observed K+ effluxes are not carried by any of the K channels observed here, but probably by electrically silent mechanisms which do not depend on the observed depolarization.
The depolarization seen in P. vulgaris is unlikely to be a component of the pathway that causes a rise in K+ efflux, since it seems both too small and too slow in onset and recovery. An increase in turgor also causes a depolarization in the turgor-regulating charophytes Chara longifolia (Bisson et al., 1995) and Lamprothamnium succinctum (Okazaki et al., 1984). In Lamprothamnium papulosum Beilby and Shepherd give evidence that the depolarization is also to values between EK and ECl and is due to the opening of both K and Cl channels (Beilby and Shepherd, 1996). It is conjectured on the other hand that the similar depolarizations seen here may result from a drop in cytoplasmic K+ concentration at a time when the PD is determined largely by K+ diffusion. If that were the case, the internal K+ concentrations would be as given in the tabulation below, which is calculated assuming an external K+ concentration of 2 mM (Walker et al., 1995):
|
These values seem biologically plausible, and their changes are consistent with loss of internal ions being a major mechanism of osmotic adjustment, as already suggested by the data on sucrose and K+ efflux (Fig. 1). This hypothesis explains the failure of the PD to return to a more negative value when adjustment is complete.
How many photosynthate efflux systems?
Large steps up in turgor can produce a large, PCMBS-insensitive, photosynthate efflux (Figs 2, 3; Patrick et al., 1986). Previous experience of PCMBS inhibiting a fraction of the normal unloading efflux suggests that this large, PCMBS-insensitive efflux is by a passive mechanism. This mechanism is not further investigated here, since the main focus is on the effects of turgor-change on the chemiosmotic system.
By contrast the effect of a more moderate increase in turgor on sucrose efflux can be partially inhibited by PCMBS (a sucrose antiport inhibitor in this system, Fieuw and Patrick, 1993), in parallel with the inhibition of the basal efflux (Table 1). This suggests that the active system is involved in responses to moderate turgor changes. This is confirmed by the inhibition of both basal and elevated efflux by EB, shown to be a pump inhibitor in this system (Walker et al., 1995), and by PZ, shown to be a sucrose transport inhibitor (Bush, 1993). This suggests that both pump and antiport are needed for a component of the response, i.e. a component that requires the operation of a coupled system.
There is a good correlation between the net H+ influx and the sucrose efflux in both species during the efflux time-course for detached seed coats (data not shown) whether exposed to a step up in turgor or not. It is not possible to measure the stoichiometric ratio of sucrose to H+ carried by the antiporter from the ratio of net H+ influx to sucrose efflux, because the (relatively small) net H+influx is the difference between two larger quantities—the H+ influx by antiport and the H+ efflux by the pump. Under the conditions of this study the imbalance of H+ fluxes would be compensated by a relatively small imbalance between K+ and anion effluxes. The net H+ influx is, as expected from this interpretation, increased by pump inhibition by EB and decreased by antiport inhibition by PZ (Table 2).
The pump rate can be estimated from the difference between sucrose efflux and H+ influx. At 20 min after the step-down in EOC the pump rate is close to 210% of control in both P. vulgaris and V. faba. This stimulation is now considered.
Under moderately elevated turgor the average cytoplasmic pH in unloading cells of P. vulgaris goes more acid by a calculated 0.45 unit (see Results). The direction of this pH change shows that a moderate step up in turgor stimulates antiport more than it stimulates the H+ pump. It also makes the membrane PD of the unloading cells more positive, supporting this interpretation.
It is then a question whether a step up in turgor stimulates the proton pump directly or through the lowered cytoplasmic pH. The drop in cytoplasmic pH of about 0.45 unit is nearly a 3-fold increase in cytoplasmic [H+]. This may well be enough to explain the observed 2-fold pump stimulation by high turgor as a kinetic effect of cytoplasmic [H+] on the pump rate. So there is no need to postulate a direct effect of turgor on H+ pump in these experiments.
These results are consistent with the view that turgor affects the antiporter only or primarily, and not consistent (at least in P. vulgaris) with the opposite alternative, that turgor affects the pump only or primarily.
There is a consensus view over a range of plant cell types that turgor change does affect the H+ pump in a different direction, i.e. that it is stimulated by lowered turgor, and that the effect is direct, not mediated by changes, for example, in cytoplasmic pH (Li and Delrot, 1987, for V. faba pod mesocarp cells; Curti et al., 1993, for Arabidopsis thaliana cultured cells of unstated origin). Kinraide and Wyse (Kinraide and Wyse, 1986, sugar beet root) suggest that raised turgor inhibits the pump, but that it is still capable of being stimulated by lowered cytoplasmic pH. Thus minor changes in the consensus scheme would accommodate the present model for the very specialized cells used here: probably no more than providing a turgor signal to the sucrose transport mechanisms—it may not even be necessary to alter very much the usual effect of turgor on the pump.
Comparing the efflux systems
The data of Table 4 allow a clear distinction to be made between the K+ and sucrose efflux systems, in spite of the apparent similarity of their responses to a turgor step-down shown in Figs 1 and 2.
No agents that alter the normal and the enhanced K+ efflux unequally have been found, with the doubtful exception of CTC. Thus they may all act on the efflux porter itself, or close to it in some normally-active control sequence, but none specifically blocks the enhancing effect of a turgor increase. So none can be thought of as helping to define the signal pathway. There is no evidence that normal and enhanced efflux occur through different porters—it is accepted for the moment that they go through the same porter, as shown in the summary diagram (Fig. 5).
|
) in the legume seed coat unloading cell membrane. Transport systems responding to increased turgor are shown as filled symbols, those not responding as open symbols. Some inhibitions and stimulations are shown which distinguish between the different transport systems.The same is not true of the agents Gd3+ and La3+ that inhibit sucrose efflux: they reduce the normal efflux but have unexpectedly little effect on the enhanced efflux, while A21387+CPA inhibits the latter and leaves the former untouched. This is only consistent with the systems carrying normal and enhanced efflux being different. Since there is evidence that both normal and enhanced efflux are sensitive to the pump inhibitor EB, and that enhanced efflux acidifies the cytoplasm, it is concluded that both are proton-coupled. It is remembered that there is previous evidence for a passive efflux enhanced by large steps down in turgor, so three different sucrose transporters are shown in the summary of this working hypothesis (Fig. 5
).
|
Conclusions |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
|---|
These results show that after a moderate step up in turgor, both H+ pump and sucrose/H+-antiport processes are stimulated. In P. vulgaris the following is observed:
- • a turgor rise increases the antiport rate, increasing H+ influx
- • this decreases the pH of the cytoplasm
- • this decrease is sufficient to cause the observed increase in the rate of the H+-pump.
In V. faba the following is observed:
- • a turgor rise increases the antiport rate, increasing H+ influx
- • the turgor-enhanced sucrose antiporter is sensitive to inhibition by A23187+CPA while the normal antiporter can be inhibited by Gd3+, La3+ and EGTA.
These results also show that after a moderate step up in turgor, K+ efflux is stimulated. In P. vulgaris the following is observed:
- • a turgor increase depolarizes the plasma membrane, but this depolarization does not directly cause the K+ efflux.
In V. faba there is thus evidence that the normal and the enhanced sucrose antiport fluxes go by different routes, while normal and enhanced K+ effluxes go through the same porter, which can be inhibited by Gd3+ and La3+ and stimulated by W-7 and CTC. This porter is not electrogenic, so it is not a KOR. The sucrose results imply that there are three parallel transporters, when the passive system is included, and that two of these are turgor-sensitive.
Figure 5 summarizes these conclusions and suggestions on the assumption that the systems in P. vulgaris and in V. faba are similar. It represents a reasonable current working hypothesis.
|
Acknowledgments |
|---|
This work was funded by an ARC Large Grant to JWP and NAW. We thank Dr Mary Beilby for helpful discussions and an anonymous referee for helpful suggestions.
|
Notes |
|---|
3 Present address: School of Biological Sciences, Flinders University of South Australia, Bedford Park, SA 5042, Australia.
4 Present address: School of Biological Sciences, University of Sydney, NSW 2006, Australia.
5 To whom correspondence should be addressed. Fax: + 61 2 9385 5981. E-mail: alanw{at}mail.usyd.edu.au
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
|---|
Beffagna N, Romani G.1988. Effects of two plasmalemma ATPase inhibitors on H+ extrusion and intracellular pH in Elodea densa leaves. Journal of Experimental Botany 39, 1033–1043.
Beilby MJ, Shepherd VA.1996. Turgor regulation in Lamprothamnium papulosum. I. I/V analysis and pharmacological dissection of the hypotonic effect. Plant, Cell and Environment 19, 837–847.
Bisson MA, Kiegle E, Black D, Kiyosawa K, Gerber N.1995. The role of calcium in turgor regulation in Chara longifolia. Plant, Cell and Environment 18, 129–137.
Briarty LG, Coult DA, Boulter D.1969. Protein bodies of developing seeds of Vicia faba. Journal of Experimental Botany 20, 358–373.
Bush DR.1993. Proton-coupled sugar and amino-acid transporters in plants. Annual Reviews of Plant Physiology and Plant Molecular Biology 44, 513–542.[Web of Science]
Cooper S, Lerner HR, Reinhold L.1991. Evidence for a highly specific K+/H+ antiporter in vesicles from oil-seed rape hypocotyls. Plant Physiology 97, 1212–1220.
Cote GG.1995. Signal transduction in leaf movement. Plant Physiology 109, 729–734.[Web of Science][Medline]
Curti G, Massardi F, Lado P.1993. Synergistic activation of plasma membrane H+-ATPase in Arabidopsis thaliana cells by turgor decrease and by fusicoccin. Physiologia Plantarum 87, 592–600.
De Jong A, Koerselman-Kooij JW, Schuurmans JAMJ, Boorstlap AC.1996. Characterization of the uptake of sucrose and glucose by isolated seed coat halves of developing pea seeds. Evidence that a sugar facilitator with diffusional kinetics is involved in seed coat unloading. Planta 199, 486–492.
Felle H.1989. K+/H+ antiport in Riccia fluitans: an alternative to the plasma membrane H+ pump for short-term pH regulation? Plant Science 61, 9–15.
Fieuw S, Patrick JW.1993. Mechanism of photosynthate efflux from Vicia faba L. seed coats. I. Tissue studies. Journal of Experimental Botany 44, 65–74.
Kinraide TB, Wyse RE.1986. Electrical evidence for turgor inhibition of proton extrusion in sugar beet taproot. Plant Physiology 82, 1148–1150.
Laver DR, Cherry CA, Walker NA.1997. The actions of calmodulin antagonists W-7 and TFP and of calcium on the gating kinetics of the calcium-activated large conductance potassium channel of the Chara protoplasmic drop: a substate-sensitive analysis. Journal of Membrane Biology 155, 263–274.[Web of Science][Medline]
Li Z-S, Delrot S.1987. Osmotic dependence of the transmembrane potential difference of broadbean mesocarp cells. Plant Physiology 84, 895–899.
MacRobbie EAC.1993. Ca2+ and cell signalling in guard cells. Cell Biology 4, 113–122.
Offler CE, Nerlich SM, Patrick JW.1989. Pathway of photosynthate transfer in the developing seed of Vicia faba L. Transfer in relation to seed anatomy. Journal of Experimental Botany 40, 769–780.
Okazaki Y, Shimmen T, Tazawa M.1984. Turgor regulation in a brackish Charophyte, Lamprothamnium succinctum. II. Changes in K+, Na+ and Cl- concentrations, membrane potential and membrane resistance during turgor regulation. Plant and Cell Physiology 25, 573–581.
Okazaki Y, Tazawa M.1990. Calcium ion and turgor regulation in plant cells. Journal of Membrane Biology 114, 189–194.[Web of Science][Medline]
Patrick JW.1984. Photosynthate unloading from seed coats of Phaseolus vulgaris L. Control by water relations. Journal of Plant Physiology 115, 297–310.
Patrick JW.1994a Turgor-dependent unloading of photosynthates from coats of developing seed of Vicia faba L. Turgor homeostasis and set-points. Physiologia Plantarum 90, 367–377.
Patrick JW.1994b. Turgor-dependent unloading of assimilates from coats of developing legume seed. Assessment of the significance of the phenomenon in the whole plant. Physiologia Plantarum 90, 645–654.
Patrick JW, Jacobs E, Offler CE, Cram WJ.1986. Photosynthate unloading from seed coats of Phaseolus vulgaris L. Nature and cellular location of turgor-sensitive unloading. Journal of experimental Botany 37, 1006–1019.
Tester M.1990. Plant ion channels: whole-cell and single-channel studies. New Phytologist 114, 305–340.[Web of Science]
Thompson LJ, Hall JL, Williams LE.1994. A study of the effects of inhibitors of the animal sarcoplasmic/endoplasmic reticulum-type calcium pumps on the primary Ca2+-ATPases of red beet. Plant Physiology 104, 1295–1300.[Abstract]
Thorpe MR, Minchin PEH, Williams JHH, Farrar JF, Tomos AD.1993. Carbon import into developing ovules of Pisum sativum. The role of water relations of the seed coat. Journal of Experimental Botany 44, 937–947.
Tsein RY.1989. Fluorescent indicators of ion concentrations. Methods in Cell Biology 30, 127–156.[Web of Science][Medline]
Walbot V, Clutter M, Sussex IM.1972. Reproductive development and embryogeny in Phaseolus. Phytomorphology 22, 50–68.
Walker NA, Patrick JW, Zhang W-H, Fieuw S.1995. Efflux of photosynthate and acid from developing seed coats of Phaseolus vulgaris L.: a chemiosmotic analysis of pump-driven efflux. Journal of Experimental Botany 46, 539–549.
Zhang W-H, Atwell BJ, Patrick JW, Walker NA.1996. Turgor-dependent efflux of assimilates from coats of developing seed of Phaseolus vulgaris L.: water relations of the cells involved in efflux. Planta 199, 25–33.
Zhang WH, Walker NA, Tyerman SD, Patrick JW.1997. Mechanism of solute efflux from seed coatscolon; whole-cell K+ currents in transfer cell protoplasts derived from coats of developing seeds of Vicia faba L. Journal of Experimental Botany 48, 1565–1572.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  W.-H. Zhang, N. A. Walker, J. W. Patrick, and S. D. Tyerman Pulsing Cl- channels in coat cells of developing bean seeds linked to hypo-osmotic turgor regulation J. Exp. Bot., May 1, 2004; 55(399): 993 - 1001. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W.-H. Zhang, M. Skerrett, N. A. Walker, J. W. Patrick, and S. D. Tyerman Nonselective Currents and Channels in Plasma Membranes of Protoplasts from Coats of Developing Seeds of Bean Plant Physiology, February 1, 2002; 128(2): 388 - 399. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. T. van Dongen, R. G.W. Laan, M. Wouterlood, and A. C. Borstlap Electrodiffusional Uptake of Organic Cations by Pea Seed Coats. Further Evidence for Poorly Selective Pores in the Plasma Membrane of Seed Coat Parenchyma Cells Plant Physiology, August 1, 2001; 126(4): 1688 - 1697. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||