4.1.3  Membrane proteins -catalysts for transport

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Some proteins involved in solute transport through plant membranes have been identified by patch clamping and, in a few cases, their genes cloned. Ion compartmentation tells us that many more await discovery in higher plants.

(a)  Passive transport systems

Proteins involved in passive transport facilitate non-energised flow of solutes and water down their respective energy gradients. Movement of a solute or water through these transport proteins is not coupled to movement of any other solute or to release of free energy from a metabolite. The proteins that facilitate passive transport are diverse; some are specific for particular ions and allow high transport rates per protein molecule (ion channels), some are specific for water (water channels or aquaporins) and some are specific for neutral solutes and may have slower transport rates per protein molecule.

Water channels or aquaporins

Some plant and animal membranse have much higher permeability to water than can be explained by diffusion rates through a lipid bilayer. Futhermore, the activation energy for diffusion of water across a plant membrane is lower than would be expected across a li[id bilayer, where water has to overcome the high-energy barrier of partitioning into a very hydrophobic oily layer (Figure 4.3). Some reports put the activation energy for water flow across membranes as low as the value for free diffusion of water. In other words, water enters the membrane about as readily as it diffuses through a solution. This suggests that water is moving across the membrane through a pathway other than the lipid, perhaps some kind of water pore or water channel. Since the discovery of water channel proteins in animal cell membranes, molecular biologists have also discovered that similar proteins exist in plants.

The approach has been to inject genetic material from plants into Xenopus oocytes (a particular type of frog’s egg). Either cDNA arising from screens of cDNA libraries can be injected into the Xenopus nucleus or poly (A)+-RNA injected into the oocyte cytoplasm where it is translated. The Xenopus oocyte is particularly useful because it is large, enabling observations of cell response to foreign proteins. It is one of several expression systems along with Chara (giant algal cells) and yeast cells. Plant water transport proteins expressed in the oocyte plasma membrane result in physiological changes; for example, the oocyte swells rapidly when the external osmotic pressure in the bathing medium is lowered (Figure 4.10a). Provided that the increase in water permeability is not a con-sequence of some other change or a side effect of other types of transport, it can be concluded that the protein catalyses transport of water across membranes. In plants, this protein could be located in the tonoplast, plasma membrane or endo-membranes. The first plant aquaporin gTIP (tonoplast integral protein) that was discovered occurs in the tonoplast and prob-ably accounts for its high water permeability. Mercuric chloride inhibits water channels, as shown by using the pressure probe in intact cells (Figure 4.10b, c).

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Figure 4.10 Evidence for presence of aquaporins (water channels) in plant membranes. (a) Rate of change in volume of Xenopus oocytes after lowering osmotic pressure of the external medium. Oocytes express foreign membrane proteins (Section 4.1.3(a)). In this case two tonoplast integral proteins (TIP proteins) were expressed in Xenopus membranes, causing a marked increase in water permeability of the oocyte and rapid swelling. ‘Controls’ were oocytes without foreign proteins and water—injected oocytes. (b) Sensitivity of two TIP proteins to mercuric chloride (HgCl2), a general inhibitor of aquaporins in animal membranes. Osmotic water permeabilities peaked at c. 20 × 10-3 cm s-1. (c) Inhibitory effect of HgCl2 on hydraulic conductivity of the freshwater alga Chara corallina. Inhibition is reversed by applying mercaptoethanol to block the action of HgCl2. Under mercury inhibition, the activation energy of water flow increases markedly indicating that water flow is now restricted to ditfusional flow across the lipid bilayer, that is, aquaporins are blocked. Hydraulic conductivity peaked at c. 5 × 10-6 m s-1 MPa-1 ((a) From Maurel et al. 1995; (b) from Daniels et al. 1996; (c) from Schutz and Tyerman 1997; reproduced with permission of (a) EMBO Journal, (b) Plant Cell, (c) Journal of Experimental Botany)

Why are there water channels in membranes when the lipid itself is already so permeable to water (Figure 4.2)? There are several rationales for the presence of water channels in plant membranes. One is that specialised transport proteins permit control of water flow. That is, a water channel protein may be turned on and off, for example by phosphorylation, while water permeability of the lipid is essentially constant. In animal cells, specifically kidney, water channels are controlled by the antidiuretic hormone. It remains to be seen if plant hormones could also influence the function of water channels.

A second rationale for the presence of water channels is to balance water flow and prevent bottlenecks. In roots, water channels are most abundant in the endodermis and inner stele where water flow across membranes is rapid. Aquaporins appear to be present at all points along roots but the possibility that they are more strongly expressed in root apices, where water flow can be rapid, remains open (Figure 3.28).

Ion channels

Following the advent of the patch clamp technique (Section 4.2.3) an explosion has occurred in identification of ion channels in animal and plant cell membranes. The plasma membrane of one plant cell can have as many as four distinct K+ channels, two types of Cl channel and a Ca2+ channel. Why are there several channel types for one ion? The answer appears to lie in: (1) the nature of control exerted on the channel and (2) the capacity of large fluxes through ion channels (> 106 ions per second) to alter membrane potential rapidly. Ion movements in cells must be regulated tightly over a wide concentration range because of the many processes that are influenced by ion activities (Section 4.1.1).

Channels open and close randomly in time under the control of gating factors. The probability that a channel is open (Popen = time open/total time) is governed by various factors including: (1) membrane voltage; (2) binding of a ligand and (3) membrane tension. Gating of ion channels by membrane tension is likely to be instrumental in the control of P. Voltage gating is particularly interesting since ion channels have a strong capacity to alter voltage, therefore influencing their own control factor. This can lead to feedback effects so that transient swings in membrane voltage are often explained by voltage-gated ion channels. When an ion channel opens, it tends to drive the membrane voltage towards the equilibrium potential for whichever ion permeates that channel; gating achieved by this voltage change can close the channel and damp further redistribution of charge.

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Figure 4.11 Transport systems in plasma membranes that are essential for volume change in guard cells driven by net flux of potassium chloride (KCl). (a) An open stoma, in which the proton pump generates a large negative membrane potential. Blue light and red light stimulate proton pumping in guard cells. Negative potential drives K+ influx through a K+ inward-rectifier channel. The pH gradient developed by proton pumping drives Cl--proton symport that must exist on the plasma membrane, although it has yet to be demonstrated. (b) A closing stoma where Ca2+ influx is a signal that initiates closure. Influx of Ca2+ depolarises the membrane (makes it less negative). An anion channel turns on, allowing Cl- to escape, further depolarising the membrane to the point that membrane potential becomes more positive than the Nernst potential for K+. The K+ outward-rectifier channel then opens, allowing K+ to leave the cell. Overall, a small amount of Ca2+ enters, followed by efflux of equal amounts of K+ and Cl-, causing an osmotically significant change.

Voltage gating can lead to ion channels behaving like nutrient valves. For example, a K+ channel that only opens when the membrane voltage is made more negative than –120 mV will tend to let only K+ move inwards, since the Nernst equilibrium potential for K+ (EK) is generally less negative than this. Rectification (i.e. only letting current pass in one direction) is a characteristic of many ion channels. This channel is called a K+ inward-rectifier and is thought to be responsible for K+ uptake from external solutions containing higher than 1 mM K+ (producing an inwardly directed electrochemical gradient of K+). It is present in root hair cells and stomatal guard cells (Figure 4.11(a) and Section 4.2.6). Other channels such as the K+ outward-rectifier are also sen-sitive to concentrations of K+ on both sides of the membrane. Combined with voltage gating, this channel becomes sensitive to EK so that it will only participate in K+ flow out of cells. The K+ outward-rectifier is probably responsible for the rapid release of K+ when guard cells lose osmotic solutes and stomata close (Figure 4.11b). It may also control release of K+ into xylem vessels.

Ligand-gated channels in plants are often either Ca2+-permeable channels or anion-permeable channels. Anion channels in the guard cell plasma membrane are gated by external malate and auxin and seem to require nucleotides (e.g. ATP) on the cytoplasmic side. They are probably involved in depolarising the guard cell which is necessary to drive efflux of K+ (Figure 4.11b) Anion channels in general are particularly effective at strongly depolarising the membrane since the resting potential is negative while the equilibrium potential for Cl is usually positive except in very saline conditions.

Channels are involved in more than transfer of nutrients such as K+ and NH4+. They can also catalyse Ca2+ release into the cytoplasm, an important event in signalling. For example, Ca2+ channels located on the tonoplast have been shown to be gated by inositol 1,4,5-trisphosphate and another by cyclic ADP-ribose. These molecules are signal transducers in animal cells and are likely to be also involved in stimulus-response coupling in plant cells (Section 9.3). In plant cells, vacuoles contain high concentrations of Ca2+ (c. 10 mM) so that when Ca2+ channels in the tonoplast open, there is a strong gradient for Ca2+ to flow into the cytoplasm, where it is scarce. A significant increase in cytosolic Ca2+ concentration results. Enzymes in the cytoplasm that are Ca2+-dependent, such as Ca2+-dependent protein kinases, then modify other enzymes by phosphorylating them in a signal transduction cascade.

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Figure 4.12   Patch clamp experiments on protoplasts isolated from the root tip of wheat. When aluminium (Al3+) is added, an anion channel opens, as seen from traces of current (Im) made over 32 min. The single channel openings can be observed (horizontal lines) in the whole-cell configuration (From Ryan et al. 1997; reproduced with permission of PNAS)

Another interesting role of ion channels in roots is to modify the concentration of ions in the surrounding solution. One example is an aluminium (Al3+) tolerance mechanism in wheat roots. Iron–aluminium complexes are a basic ingredient of clay, Al3+ being released from these complexes when soil becomes acidic (Section 16.5). Even very low ionised Al3+ concentrations (less than 10 µM) are toxic to root tip cells. In some tolerant varieties of wheat, and other species, organic anions (mainly malate in wheat) are released from root tips when tip cells register the presence of free Al3+. Malate and citrate bind very strongly to Al3+, thereby protecting the root membranes from contact with Al3+. Significantly, there is an anion channel in root tip cells that is not evident in mature root tissues and this channel is specifically opened even at low concentrations of Al3+ (but not other trivalent cations) (Figure 4.12). Features of the anion channel measured in patch clamp experiments match many of the characteristics of malate exudation from intact root tips exposed to Al3+. This channel may therefore be one of the gene products that confers tolerance in wheat. However, questions remain as to how the presence of Al3+ turns on the channel; are there specific receptors for Al3+ in membranes closely linked with the channel or do Al3+ receptors control the channels via a signal cascade?

Several genes that encode ion channel proteins have now been cloned and studied in expression systems such as the Xenopus oocyte. Not surprisingly these genes show some homology with animal channel counterparts and in some instances the plant genes were found by using probes based on this sequence homology (e.g. the voltage-dependent Cl channel from tobacco). In the case of KAT1, which appears to be primarily expressed in guard cells, there is homology with the SHAKER genes of Drosophila. However, in Drosophila these channel-encoding genes code for outward-rectifier channels, posing interesting questions as to what components of the protein cause the channel to have an opposite voltage dependency in plants. Using site-directed mutagenesis so that amino acid sequence can be altered in the protein, and studying the altered properties of the channel in an expression
system, it will be possible to determine the regions of the protein responsible for selectivity and voltage sensitivity. It may even be possible to alter the properties of channels so that they become more selective for particular ions.

(b)  Some key active transporters

Plasma-membrane-associated H+-ATPases are crucial to plant cell function but are low-abundance proteins representing at most 1% of plasma membrane protein and 0.03% of total cellular protein. These proteins play a primary role in cells by pumping protons and thereby generating a protonmotive force which drives secondary active transport. Plasma membrane H+-ATPase has a phosphorylated intermediate during the catalytic cycle which places it in the class of P-type ATPases: this class includes a variety of cation pumps such as the Ca2+ pump. Na+–K+ ATPases, which are the primary active transport systems in most animal cells, are also P-type ATPases and share some homology with plant and fungal H+-ATPases. The H+-ATPase protein has a molecular weight of 100 kD and is thought to function as a monomer in vivo. In Arabidopsis thaliana there are at least 10 separate genes coding for different isoforms of H+-ATPase, probably corresponding to different cell and tissue types and to different developmental stages. For example, a H+-ATPase responsible for energising sucrose transport has been identified in developing legume seeds (Figure 5.32). Variations in affinities of ATPases for ATP might reflect functional requirements of different isoforms.

Fine control of H+-ATPase activity is achieved by a variety of factors, commensurate with the protein’s pivotal role in plasma membrane transport. Cytoplasmic pH is closely regulated in plant cells and the H+ pump has a central role in achieving this homeostasis. Plant hormones such as auxins stimulate H+-ATPases and the resulting acidification of cell walls is thought to be a primary step in cell wall loosening to allow expansion growth. Auxin also seems to increase H+-ATPase incorporation into membranes, probably by activating genes involved in synthesis or incorporation of protein into membranes. Fungal elicitors and toxins (e.g. fusicoccin) also stimulate H+-ATPase and these have been used in many studies on regulation of the pump. Specific receptors for these molecules on the plasma membrane appear to set off a signalling response capable of stimulating H+ pumping. Secondary messenger pathways in the cytoplasm and plasma membrane involving G-proteins and protein phosphorylation/dephosphorylation are involved in signal amplification, probably through a Ca2+-dependent cascade. In this case, control of the pump resides partly in an autoinhibitory domain on the carboxy terminus of the protein because trypsin digestion of this domain (or engineering coding errors into the gene) results in increased activity.

The plasma membrane H+-ATPase has been purified and incorporated into an artificial lipid bilayer. This has allowed coupling between ATP hydrolysis and pumping of H+ to be measured electrically under conditions where the energy supply and gradient can be rigorously controlled. Such experi-ments on the isolated pump as opposed to in vivo studies where other transport systems interfere should yield unprecedented detail of the pump mechanism particularly if it is combined with site-directed mutagenesis to alter some key amino acids in the protein.

Various types of sugar, amino acid and peptide and amine transporters have been identified in plants, originally via classic biochemical and biophysical techniques and more recently by molecular approaches. Various amino acid transporters have been identified in plants and several genes have been cloned. These genes are not all from one gene family and the transporters they encode show differences in specificity to amino acids. The sugar transporters are vitally important for redistribution of assimilates to non-green parts of the plant and to developing seeds. The mechanism of long-distance transport of assimilates in the phloem is ultimately driven by plasma membrane H+-ATPase, developing voltage and H+ gradients capable of energising symport or antiport of sugars (Section 5.6). One of the sucrose transporter genes is expressed in phloem, either in the companion cell plasma membrane or sieve tube membranes. Another sucrose transporter gene is expressed during seed development. Specialised plant tissues are likely to have assimilate demands which differ from those cells surrounding them and therefore we might expect further progress in the identification of tissue-specific transporters. Vascular tissues, which exhibit substantial cell to cell variations in assimilate fluxes, are candidates for such an analysis.

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