CASE STUDY 4.1  The power of biological pumps

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R. L. Bieleski

Early studies on transport processes in plants explored the general features of mineral nutrient uptake. When a pump works against a gradient, energy has to be used; and so it came as no surprise that anything which blocked energy-generating pathways (poisons such as cyanide, azide or dinitrophenol) or slowed metabolism (cold temperature, anaerobiosis) interfered with uptake. Once ions were inside cells, they did not diffuse readily back into the surrounding medium, even when energy supply was virtually halted by these methods; that is, the pumps had non-return valves. Though ions did not move back into the medium, they were readily transferred to other cells and eventually to the body of the plant. Our ability to explore pump behaviour took a great leap forward once radiotracers became available to study the mechanisms involved. Over the last 50 years or so, our concepts of ‘how pumps work’ have developed and changed, and Australasian scientists have been strong contributors to the learning process. In the early 1950s, a group in the CSIRO led by R. N. Robertson pioneered the use of concepts and methods taken from physics to attack this biological problem, and in so doing helped found the discipline of biophysics. Based on their use of carrot slices and the chloride (Cl) ion as a model, they interpreted uptake as an essentially ‘electrical’ phenomenon, with ion movement being directly coupled with flow of electrons through the respiratory cytochrome pathway.

Various observations soon began accumulating which called for a rethink. In a nutshell, pumps operated on too many dif-ferent molecules and had too many biological properties to be run in that way. For example, when working in Robertson’s laboratory, I showed that glucose and sucrose movement into sugar cane slices also satisfied the criteria of pump-driven processes (e.g. with selectivity and movement against a concen-tration gradient), like Cl transport, yet the sugar molecule was non-ionic and should have been inaccessible to electrical systems. A possible option was that sucrose was made into a charged form (e.g. by phosphorylation), but other evidence was pushing us more towards modelling uptake on the behaviour of enzymes.

Anaylsis of pumps with an enzyme analogy

Why was an individual pump so selective, with methyl glucose behaving very differently from glucose, with nitrite and nitrate having totally separate uptake systems, and with orthophosphate (HPO42–) appearing to share its pump with arsenate (HAsO42–) but not with sulphate (SO42–)? The enzyme-like characteristics of potassium (K+) uptake were demonstrated by E. Epstein (University of California) using barley roots (Figure 4.16). This enzyme analogy was fruitful because uptake rates plotted against concentration fitted a rectangular hyperbola allowing a ‘double-reciprocal’ or ‘Lineweaver–Burke’ analysis of uptake. For a single hyperbola, a plot of (1/rate) against (1/concentration) gives a straight line which reveals Vmax (‘capacity’ of the pump, its maximum rate of uptake) and Km (the sucking power or ‘affinity’ of the pump for its supply material). Competition between K+ and Rb+ is evident as competitive inhibition of Rb+ uptake in response to additions of K+ (Figure 1), a phenomenon first identified in enzyme kinetics. Even uptake of sugars into sugar cane closely followed this enzyme-like pattern, with a Km (250 µM for sucrose) of the same order as that being found for various cations. We still use these kinetic terms today and though kinetic parameters are mathematical abstractions as they stand, they took on more meaning when it was realised that Km often resembles the concentration of that ion found in typical soil solutions, while Vmax is comparable to the rate of supply needed by the plant to support its maximum growth rate.

Like enzymes, pumps can change their activity in response to presence or absence of substrates. Section 4.2.7(a) describes how this occurs for K+ and orthophosphate which allosterically regulate their carriers according to nutrient demand. Nitrogen uptake illustrates how the full range of pump activities is coordinated to optimise supply of such an important inorganic resource. Ammonium (NH4+), the favoured nitrogen source in plants, is taken up by a constitutive transport system (i.e. uptake capacity is always present). If NH4+ fulfils the nitrogen demand of a plant, a nitrate (NO3) pump is not needed so NO3-transporting capability drops to a low level. If this plant is then supplied NO3 and not NH4+, NO3-carrier activity is rapidly re-formed (induced) to sustain nitrogen uptake. Nitrite (NO2), a third but less desirable nitrogen source, can induce formation of an NO2-specific pump. Such inducible pumps are sometimes called permeases.

These ‘electrical’ and ‘enzyme’ concepts have begun to merge. Uptake is now seen as powered by the respiration path but with proton flow rather than electron flow providing the driving force, while solute entry itself is controlled and given specificity by enzyme-like transporter proteins subject to the same sort of expression controls as other gene products. These proteins have a central hydrophobic portion which sits in the lipoidal cell membrane, with two hydrophilic ends that inter-face with the aqueous apoplasm and cytoplasm at the outer and inner membrane surfaces respectively. According to one model, 5 to 12 of these proteins join in a ring to make a tube with a central channel crossing the membrane, through which transported molecules pass. The energy required to push materials against a concentration gradient is met by an ATP-driven proton pump which shifts protons (one H+ per ATP hydrolysed) from inside to outside the membrane; then as H+ returns across the membrane, its inward movement is tightly coupled with that of the material being pumped, so providing the necessary thrust in a symport or coport process.

Pumping orthophosphate

My own research interest has centred around two particular pumps, one for uncharged sugars (particularly sucrose) and one for the anion orthophosphate. They represent the extremes of what is required from a pumping system. Of all the solutes in plant cells, sugars are typically present in greatest concen-tration, and have to be moved from cell to cell in the greatest amount — in enzyme terms, the sugar pumps have to have a very high Vmax. In contrast, an orthophosphate pump must achieve a 10 000-fold concentration of a scarce resource, similar to that between the atmosphere and a high vacuum chamber of a freeze-drier. This requires a ‘high-affinity’ pump (a very low Km). Because of its extreme nature, orthophosphate shows some transport phenomena particularly well when used as a model system. For example, beetroot slices that have been pretreated by aerating in 1 mM CaSO4 at room temperature for 24 h can demonstrate some basic ortho-phosphate transport features in a three-hour laboratory session.

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Figure 1 Effect of rubidium concentration on rubidium uptake rate into barley roots, expressed in a standard plot (a), and as a double-reciprocal plot (b). In the double-reciprocal plot, the intercept on the y-axis gives (1/ Vmax), and the intercept on the x-axis gives (-1/Km).The three lines illustrate the competitive inhibition of rubidium uptake by potassium (Epstein and Hagen, 1952; reproduced with permission ofthe American Society of Plant Physiologists)

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Figure 2   Coexistence of low-affinity and high-affinity orthophosphate pumps in celery vascular bundles. Freshly excised bundles have only one pump. Curve 1 is the calculated line for a Km of 75 mM and a Vmax of 9500 nmol g-1 fresh mass h-1, and it gives an excellent fit with the experimental points (Δ) for that tissue. When the excised bundles are aerated for 24 h in 0.1 mM CaSO4 (aged), they develop a second orthophosphate pump which adds on to the first. It has the constants Km = 58 µM and Vmax = 355 nmol g-1 h-1, and is shown by curve 2. Total orthophosphate uptake in aged bundles (O) is the sum of uptake by the two pumps (curve 1 + curve 2 = curve 3) (Bieleski 1966; reproduced with permission of the American Society of Plant Physiologists)

Interpretation of orthophosphate transport has, however, presented one problem, in that the uptake rate/concentration curve often fails to conform to the simple ‘enzyme’ relation-ship of a rectangular hyperbola (Figure 1). Various inter-pretations have been made, but the most common is one I have been partly responsible for developing, in which we see the relationship arising from the joint operation of two enzyme-like orthophosphate pumps having distinct kinetic characteristics (Figure 2). We call these the ‘high-affinity’ and ‘low-affinity’ systems, where the ‘high-affinity’ system (the one that is good at scavenging phosphate from the environment) has a very low Km, around 2–5 µM orthophosphate, and the ‘low-affinity’ system has a Km of 300–800 µM orthophosphate. Leading on from this, there has been a debate about whether the two systems coexist in the plasma membrane (the view of E. Epstein) or whether we are seeing the contrasting behaviour of two different membranes in series (plasma membrane and tonoplast; G. Laties) or different cell types within the experi-mental material (M. Pitman and others). Subsequent research findings support all three concepts as companions rather than competitors. The original two-system patterns were probably the product of two carriers located in the plasma membrane. However, patch clamping now provides evidence that the tonoplast as well as the plasma membrane has a very effective phosphate transporter (Section 4.2). Similarly, studies by molecular biologists, particularly with Arabidopsis mutants as a model, confirm the coexistence of separately coded ‘low-affinity, and ‘high-affinity’ orthophosphate uptake systems; but they also imply additional orthophosphate transport systems concerned with unloading and redistribution of phosphate around the plant, and which are expressed in specific tissues. In my view, the joint presence of a ‘low-affinity’ and ‘high-affinity’ system allows plants to cope with an extremely wide range of orthophosphate concentrations. Though the soil con--cen-tration encountered by the plasma membrane of root cells is around 1–5 µM, the concentrations inside the cell confronting the tonoplast are around 5000 µM. Furthermore, if a cell unloads its orthophosphate or dies, and releases its solutes into the fluids of the intercellular system (the apoplasm), it could potentially expose the plasma membrane of adjacent cells to equally high orthophosphate concentrations. Having two systems adapted for different ends of the concentration range may give cells a better ability to manage orthophosphate uptake and redistribution than by using one system alone. Isolation of phosphate transport mutants should help a great deal in unravelling questions about the interplay of the two systems.

In summary, a plant controls orthophosphate entry by managing two inward pump systems which adjust in response to external supply (Section 4.2.7(a)). However, situations may arise where there are major and rapid changes in demand for orthophosphate inside the plant under conditions where the external supply has not changed (for example, resulting from a period of darkness or a sudden drop in nitrogen supply). If a plant were able to modulate orthophosphate loss actively through the efflux mechanism as well as orthophosphate uptake itself, a much more effective total control of net orthophosphate uptake would be achieved. Efflux may be a plant’s safety valve for orthophosphate. There is plenty of territory here for future research, but it is my guess that efflux will turn out to be part of the mechanism for maintaining the orthophosphate balance of the plant (homeostasis), with cytoplasmic orthophosphate concentration being the signaller and controller of its operation.

The concept of active, outwardly facing pumps pushing things out of a cell into the outside world has been undervalued. People interested in phloem transport (particularly of sugars) have been half-way there in talking of ‘unloading’, where move-ment is from the cell into the apoplasm around it. Mostly, the possible involvement of pumps in such ‘unloading’ is not discussed because the concentration gradient favours passive (diffusive) movement anyway. However, as the ortho-phosphate story shows, pumps may sometimes be needed to provide control of flow rather than to overcome a gradient. It is something of an irony that the most visible and common example of sugar unloading we have, the secretion of nectar by a flower nectary, almost certainly has to involve an outward-facing pump, in that the final ‘sink’ concentration is extremely high, sometimes exceeding 250 mg sugar mL–1 (about 1 molar), so that secretion must be against a concentration gradient.

Most of this case study has been about orthophosphate pumps, with their ability to overcome steep gradients. But in finishing, it is worth looking at the mass of material a sugar transporter must move. A tissue which is actively transporting sucrose (e.g. excised phloem tissue obtained as stripped celery vascular bundles) can move up to 2 mg sucrose g–1 fresh mass h–1. That corresponds to 3 ¥ 108 molecules of sugar per cell per second, when each molecule has to be handled individually by a transporter assembly! Each transporter assembly is about 30 Å across, so that even if 10% of the membrane surface were occupied by sucrose transporter sites, the transport rate would be about one molecule of sucrose per transporter site per second. Powerful pumps indeed!

References

Bieleski, R.L. (1966). ‘Accumulation of phosphate, sulphate and sucrose by excised phloem tissues’, Plant Physiology,
41, 447–454.

Epstein, E. and Hagen, C.E. (1952). ‘A kinetic study of the absorption of alkali cations by barley roots’, Plant Physiology, 27, 457–474.

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