Early workers believed that inorganic salts were passively carried into the plant through diffusion with the absorption of water and further assumed that the translocation of the absorbed salts to different parts of the plant was dependent on transpiration.
It is known that certain ions may attain a higher concentration in the cell sap of plant tissues than in the circumambient solution. In alga Nitella for example both anions and cations accumulate in a concentration much more than the circumambient solution.
The accumulation of such ions in plant tissues in quantity many times greater than the surrounding external medium is possible only through diffusion against a concentration gradient. This phenomenon does not operate if the plant tissues are already in excess of those ions.
Therefore, it is dependent on the metabolic status of absorbing cells or tissues in plants. This phenomenon of salt accumulation is mostly prevalent in meristematic cells and decreases with the increase in cell maturity. This led to the understanding of the mechanism of salt accumulation in cells and tissues of plants, greater than the circumambient solution, defining diffusion against concentration gradient.
The high rate of respiration in the meristematic cells and its low rate in the maturing cells indicate the involvement of energy in the diffusion of ions against the concentration gradient. This explains the accumulation of salts as an active process.
Driving Forces:
The driving forces for solute transport at membrane level in plants are same as those in the other biological membrane systems. However, features of plant cell like the presence of cell wall and vacuole considerably aid the technical problems of measuring various parameters.
The driving force for the uncharged solutes is the gradient of chemical potential only while for the ions driving force has additional component due to the potential differences (p.d). If the activity of ion is known on one side of a membrane, it is possible to determine the magnitude of p.d. which will maintain equilibrium across the membrane.
This is given by Nernst equation:
Here EN is the Nernst potential (in mV) for ion species j, R the gas constant, T the absolute temperature, Zj the valency of ion, F, the Faraday constant and cj/Cji the ratio of concentration.
The Nernst potential may be compared with the measured potential across the membrane Em. If Em = ENj, a passive equilibrium situation exists and if these are different, then the energy must be expanded to maintain an equilibrium.
The Nernst equation is possible only when ions are in flux equilibrium condition across the membranes.
For non-equilibrium conditions, the active transport may be identified by the flux ratio for an ionic species across the membrane.
The relationship between the influx and efflux, ratio is given by Ussing- Torell equation:
where φjoi is influx and φjio is efflux. When the measured flux ratio differs from the predicted value, ion species is not moving passively and active transport is indicated. But this tells us nothing directly of the transport of ions across the membrane.
If the active transport involves the movement of a charged ion-carrier complex across the membrane, then it is termed as electrogenic process.
EM = Eeq + Ex
where EM is potential across the membrane, Eeq a diffusion potential and Ex an additive electrogenic mechanism.
Evidence of electrogenocity is provided by the very rapid depolarization of a membrane with metabolic inhibitors which often reduce the value of Em.
Measurement of electric conductance of plant cell membranes can provide information on their structure and organization and the manner in which ions cross them. When ion species is at electrochemical potential equilibrium, a flux φj, will contribute partial ion conductance gj, such that;
Carriers and Pumps:
Due to the low partition coefficient Kj of polar solutes, which reflects the high lipid content of cell membranes, the permeability of biological membranes to solute molecules Pj is known to decrease with increasing polarity of the permeant. For the movement of solutes, particularly ions, some form of carrier molecule is envisaged which is soluble in the membrane.
This carrier combines with permeant and transports it across the membrane. If such movement is down the gradient of chemical or electrochemical potential, the carrier process may be regarded as a facilitated diffusion and carrier may be positive. When solute movement is against potential gradient an active process is involved.
It has been observed by large number of investigators that uptake of ions by plant tissues appears to follow a relationship analogous to Michaelis-Menton equation used in studying the kinetics of enzyme reactions. Assuming that ion S binds reversibly to specific carrier C, in the membrane, the proposed model is;
where k1, k-1, k2, k-2 are the rate constants of reactions. Assuming that there is little or no counter flow from inside to outside of the membrane, the velocity of ion uptake v at ion concentration (S) in the external medium is given by
where Vmax is the maximum rate of uptake when carrier is saturated, KS is a constant characteristic of a particular ion crossing a specific membrane and is expressed in units of concentration (mol m−3).
The reciprocal of the above equation gives a linear relationship.
This enables values for Vmax and Ks to be calculated and thus affinity of a carrier for an ion may be characterized. In plant tissues, uptake of an ion often yields two or more lines when above relationship is plotted, from which different Ks and Vmax values may be obtained.
This has been interpreted as evidence for the existence of separate carriers within the membrane each with a different affinity for a particular ion. Two such carriers have been proposed for a particular ionic species; the one functional at low concentration (< 0.5 mol m−3) with a low Ks has high affinity and is termed system I, the other is functional at higher concentrations (> 0.5 mol m−3) with a higher Ks and lower affinity, and termed system II.
The location of these two uptake systems is being debated; some investigators suggest that system I is on plasma membrane, and system II is on the tonoplast, whereas others suggest that both the mechanisms are located on the plasma membrane arranged in parallel fashion (Fig. 10-1 A).
Alternate models have been proposed to explain dual isotherms without considering the concept of two or more carrier sites. Investigation of the plasma membrane on which ions are bound selectively, has been considered and dual isotherms are functional in terms of delivery of ions to different cytoplasmic compartments.
Some investigators believe that ion absorption isotherms which are the result of active processes do not always follow Michaelis-Menton formalism and passive ion movement and particularly if it is carrier mediated. The above carrier model differs from the ‘pumps’ which have been proposed to operate in maintaining ionic composition within plant cells.
In carrier model all movement of ions into and out of cell is mediated by carrier, whereas ‘pumps’ are proposed to counteract a passive ion movement or leakage in opposite direction. These pumps are located at the plasma membrane and are responsible for K+/Na+ ratio being considerably higher in the cytoplasm than in external medium of plant cells.
Passive Absorption:
The concept of outer space (free space or diffusion space):
If only a portion of the tissue volume is open to free diffusion of ions, the ions move freely in or out of the tissue. Further, the part of the tissue undergoing free diffusion will reach equilibrium with the external solution.
Consequently, the ion concentration of this part will be the same as that of external solution. The part of a cell or tissue which allows free diffusion of ions is called free space or outer space. This is true only if the entry of solute into the tissue is purely by diffusion.
It is possible to calculate the volume of “outer space” when a tissue is immersed in a solution of known concentration and allowed to attain equilibrium. The amount of salt taken up is determined. Using metabolic inhibitors or by administrating low temperature, it is possible to inhibit active absorption.
The solute quantity inside the tissue is determined and assuming that its concentration is the same as in the surrounding solution, the volume it occupies can be calculated.
Vd =A/C
where Vd = volume within the tissue available for diffusion.
A = Amount of solute absorbed
C = Concentration of solute per unit volume
Expressing this volume as a percentage of the total tissue volume (Vt) and taking this percentage as the apparent free space (Sd), following relationship is deduced:
Sd = 100 Vd/ Vt= 100 A/ Vt C
Apparently, the free space is that portion of the volume of tissue which can be in diffusion equilibrium with an outside medium.
It may or may not be exactly equal to the actual free space or “outer space”. The apparent free space probably consists of cell wall, intercellular spaces (Levitt, 1957) or even part of cytoplasm.
Bean root tips, immersed in a KCI solution, reach equilibrium in 20 min in the absence of metabolic energy.
Further, the volume of the tissue involved, is calculated. The concentration of KCI in the external solution when increased, the volume of tissue allowing free diffusion also increases.
Since the active transport was inhibited it is concluded that a passive accumulation of ions against a concentration gradient must have taken place.
The accumulation of ions against a concentrating gradient did not involve participation of metabolic energy but was accomplished through ion exchange mechanism and through the establishment of Donnanequilibria.
Ion Exchange:
Ions are absorbed through the surfaces of the cell wall or membranes of cell exchange with ions of equivalent charge from the external solution.
A similar ion exchange occurs between the soil solution and colloids. Sometimes, in the uptake of nutrient cations from a solution through roots exceeds than the anions, while with other solutions, the reverse is true. In these instances neutrality is maintained.
Firstly, if uptake of nutrient on ions exceeds cation uptake, hydroxyl and bicarbonate ions are transported outwardly from inside the cell. If cation absorption is rapid than the anions, some hydrogen ions are exchanged by cells. Secondly, excess of cations uptake is accompanied by the simultaneous bicarbonate absorption and hydroxyl ions, while excess anions may be taken up with hydrogen ions.
H+ and OH– ions are formed by the dissociation of water, and H+ and HCO–3 by the dissociation of H2CO3.
The ionic exchange process is explained as follows:
a. Carbonic Acid Exchange Theory:
CO2 from respiration is continuously released at the root tip. It combines with the water to form carbonic acid (H2CO3) and the latter dissociates into H+ and HCO3– ions. The H+ ions exchanges with cations are absorbed on the clay micelle and enter the soil solution and diffuse to the root surface.
A zone of carbonic acid is developed in the root tip because of high respiratory activity. The H+ ions get absorbed from the root surface, and may exchange with cations in the soil solution released from the clay micelle. The cation may also pair with HCO3− or OH− ions and absorbed together (Fig. 10-2).
b. Contact Exchange Theory:
A similar exchange of ions may take place between the root and clay micelle at the point where they are in direct contact with each other without being first dissociated in the soil solution.
Donnan Equilibrium:
This theory takes into account the effect of fixed or indifussible ions. A differentially permeable membrane separates cell from the external solution. The cell has a concentration of anions to which the membrane is impermeable.
If a membrane which is freely permeable to the cations and anions present in the external medium is used, an equal number of cations and aninos will diffuse across the membrane into the cell till equilibrium is reached.
However, additional cations will be required to balance the negative charge of the “fixed” anions on the inside of the membrane (Fig. 10-3). Therefore, the cation concentration would be greater in the internal than in the external solution. Also, because of the excess of negative charges due to “fixed” anions, the concentration of anions in the internal solution will be less than that of the external one.
Mass Flow Hypothesis:
Many workers have proposed that ions move through root along with the stream of water under the influence of transpiration. In detopped tomato plants an increase in transpiration increases salt absorption indirectly by removing ions after they have been released into the xylem duct. The dilution thus caused promoted ion absorption.
There is a good possibility to assume that a part of the total salt uptake, by paint may result from passive absorption as follows:
(i) Free diffusion of ions along concentration gradient into the apparent free space of a tissue.
(ii) Accumulation of ions against concentration gradient due to ion exchange or Donnan equilibrium, and
(iii) Lastly, mass flow of ions through roots due to transpirational “pull’ may also occur. In all these processes, no metabolic energy is involved.
Active Transport:
In several instances there is extensive uptake of nutrients and this uptake is brought about against a chemical or an electrical potential gradient. Such an uptake necessarily compels one to assume the functioning of an active process.
Analysis of the ion accumulation in the sap of Nitella and Valonia reveals that potassium and certain other ions are commonly present in higher concentrations inside the plant cells than in the environments.
However, sodium and calcium may or may not be accumulated. If in a cell, molecules have to be transported against a concentration gradient, it must utilize energy. Transport against such a gradient is referred to as active uptake.
Whatever the mechanisms suggested so far for explaining active transport all assume active transport of an ion across an impermeable membrane and that such a transport is accomplished through the agency of a carrier compound situated in the membrane.
Electrogenic Pump:
In a tissue or a cell there is “inner space” through which ions penetrate with the help of metabolic energy. Inner spaces comprise vacuoles and protoplasts of all cells of a tissue, interconnected by plasmodesmata.
The area between outer and inner space is impermeable to free ions. Passage across this area is thought to require the aid of specific carriers, which combine with ions in outer space and release them in inner space (Fig. 10-4).
This impermeable barrier is referred to as a membrane and assumingly carriers exist within it. The carrier theory envisages the formation of an intermediate carrier-ion complex which has the capability to move across the above mentioned impermeable membrane.
Ions released into the inner space cannot move out and are thus accumulated. During the attachment of ion to the carrier molecule, metabolic energy is expended. Carrier concept is suggested by evidences from isotopic exchange, saturation effects and specificity.
These are described below:
Isotopic Exchange:
If tissues which were initially immersed in K2SO4 with radioactive SO4-2. Ion solution and which were allowed to accumulate labelled SO4-2 ion, are then transferred to distilled water, the ions absorbed passively leak out and those absorbed actively in the vacuoles do not leak out as long as the cells remain healthy.
If such tissues are now immersed in CaSO4 solution, the SO4−2 ions will not exchange with isotopic SO4-2 ions in the external medium. Since ions are absorbed, their movement across the membrane must be due to intervention of carriers.
Saturation Effects:
The rate of accumulation increases with the concentration of the ions in the medium up to a specific point after which further increase in concentration does not result in additional increase in the rate of accumulation. In other words, when all the active sites on the carriers are occupied, a saturation point is reached. Obviously, carrier molecules are utilized in the accumulation process.
Specificity:
The fact that rate of absorption of different ions differs and that the levels of accumulation in the root tissue differs, points towards the presence of specific carriers. Thus, potassium or cesium ions inhibit the uptake of rubidium but sodium or lithium does not affect the process. On the other hand, bromide and iodide ions inhibit chloride absorption.
Certain ions compete for specific sites on the carrier. In general, two important mechanisms of salt absorption based on the carrier concept are accepted, of these one is cytochrome pump while the other involves ATP (Fig. 10-4A).
Cytochrome Pump:
Lundegardh observed a quantitative relationship between anion absorption and “anion” or “salt” respiration but no such correlation with cation absorption existed. Addition of salt solution to respiring tissue increased their respiration rate. This was called salt respiration.
It was also noted that salt respiration and anion absorption were inhibited by cyanide or even carbon monoxide.
Based on these observations, Lundegardh advanced a theory with the following assumptions:
i. Absorption of anion is independent of cation; only anions are actively transported through the mediation of cytochrome system. The latter acts as an anion carrier.
ii. Cations move passively to balance the electrical difference caused by the accumulation of anions on the inner surface.
iii. An oxygen concentration exists from the outer to the inner face of a membrane and this favours oxidation at the outer and reduction at the inner face respectively.
As will be observed from Fig. 10-5 dehydrogenase reactions on the inner surface produce protons (H+) and electrons (e–). The electrons move out via cytochrome chain, while anions move inward.
At the outer face of the membrane the cytochrome reduced ion is oxidized losing an electron and picks up an anion.
The electron liberated unites with a proton (H+) and oxygen to form water. At the inner face the oxidized cytochrome ion becomes reduced by the electron released in a dehydrogenase reaction. The anion is released on the inside in this last reaction.
This theory has been criticised on three grounds:
i. Robertson (1951) found that DNP, an inhibitor of oxidative phosphorylation, increased respiration but decreased salt respiration indicating role of phosphorylation in ion accumulation.
ii. Only anions are capable of stimulating respiration as suggested in the theory. However, Overstreet and Handley (1955) observed that both sodium and potassium cations stimulated respiration.
iii. According to the theory, there is only one carrier for the anions which suggests competition between anions for binding sites. However, sulphate, nitrate and phosphate do not compete with one another.
The original hyphothesis of Lundegardh has been modified by several workers.
A mechanism has been suggested by Bennett Clark, whereby the lipid substance lecithin (Fig. 10-6) is suggested to absorb an ion, transports it as a complex across the membrane and then it is hydrolysed.
The ion is released inside upon hydrolysis of lecithin and the resynthesis of lecithin requires ATP.
Using Nernst equation it is possible to determine whether each different kind of ion has been taken through active or passive mechanisms. One common feature emerges i.e. involvement of some carrier system to assist the ion to cross the membrane.
In the following a brief sequence of events is proposed:
(i) Any ion which carried an electric charge will be associated with a covering of water.
(ii) The ion possibly attaches to molecular body of the membrane and its charge is neutralised. Thus, it is able to penetrate the lipophilic membrane.
(iii) Some carrier transports the ions through the membrane and releases them on the other side.
In the above, a possibility of uptake of ions is suggested but the problem does not end here. We do not have the complete information how the solutes reach the vessels of the xylem.
It is not vivid whether nutrients pass from cell to cell crossing cytoplasm-vacuole-cytoplasm way, or move passively through the cell wall or move actively through plasmodesmata.
Recently, in a review Pate and Gunning (1972) have proposed translocation of solutes through special cells with the well-defined convolutions in their internal membranes.
These cells have been named as transfer cells. These cells are especially present in the areas which are concerned with rapid lateral movement of solutes (Fig. 10-7).
Solutes may also move through a membrane by the formation of bubbles or vesicles on one side of the membrane and thus discharge their contents to the other side. This is process of pinocytosis. This is a non-selective process since the solutes are moved only as part of the small vacuole of solution and not independently. Active transport, on the other hand, is highly selective.
There is a good possibility to believe that Casparian bands regulate the path of solutes through the cytoplasm of the endodermal cells. There is also a possibility to believe that vacuoles may not be involved in transport of salts but may act as reservoirs for excess ions.
Even then the precise mechanism of discharge of solute in the xylem is not clear. One good assumption is that reduction in the oxygen supply causes the ions to leak into the xylem elements. It is well known that under conditions of poor aeration, with reduced supply of energy, all membranes show the tendency to leak.