Plant Physiology and Metabolism PDF

BBYCT-137 PLANT PHYSIOLOGY Indira Gandhi AND METABOLISM National Open University School of Sciences VOL 1 PLANT PHYSIOLOGY AND METABOLISM BLOCK 1 WATER AND MINERAL NUTRITION 5 BLOCK 2 PHOTOSYNTH...

BBYCT-137 PLANT PHYSIOLOGY Indira Gandhi AND METABOLISM National Open University School of Sciences VOL 1 PLANT PHYSIOLOGY AND METABOLISM BLOCK 1 WATER AND MINERAL NUTRITION 5 BLOCK 2 PHOTOSYNTHESIS AND TRANSLOCATION OF PHOTOSYNTHATES 121  PLANT PHYSIOLOGY AND METABOLISM Plant Physiology Course has two volumes. Volume 1 contains Block 1and Block 2 while Volume 2 contains Block 3 and Block 4. Physiology is the study of the functions of living organisms and their constituent parts. You will study the physical and chemical terms, the mechanisms that operate in living organisms at all levels, ranging from the subcellular to the integrated whole organism. Although there is striking similarity between the molecular mechanisms of plants and animals in terms of biosynthesis and catabolic pathways, and many cellular functions among the lower levels of organization, the differences are more pronounced between the higher plants and higher animals. For example plants have evolved unique machinery for synthesizing their food by photosynthesis. Although plants do not have an active pump like a heart, they possess an intricate transport system to carry water, dissolved minerals, photosynthates and hormones to all parts of the body. The respiratory mechanism is also similar at the cellular level, though there are distinctly different systems of gaseous exchange in both the groups. Plants also respond to the external stimuli, but the manifestation of their response differs from those in animals. Growth and development in plants is regulated by various external (environmental) and internal (hormonal) factors. Plants also respond to these environmental factors. The basic theme of Plant Physiology is divided into four blocks. The first block deals with the plant water relations at the cellular and whole plant level. In addition, the inorganic nutrients required by the plants, their occurrence, uptake and transport is also described. The second block comprises the energy conservation by plants through photosynthesis and translocation of the photosynthate by phloem. The third block deals with the biocatalysts, their kinetics and mechanism of action. Mechanisms operative for the oxidation of photoassimilates and other reserve food materials have been described in units on respiration. The final (fourth) block (Units 13 and 14) is devoted to a study of nitrogen assimilation by plants including the biological nitrogen fixation carried out by a few ‘gifted’ prokaryotes. Unit 15 deals with a study of plants growth regulators, their discovery, structure, mode of action and commercial application. The final Unit 16 gives a comprehensive account of the responses of plants to environmental stress highlighting various physiological adaptations for acclimation. Objectives After studying this course, you will be able to:
appreciate the water relations of the plants, mechanism of absorption of water, and the different pathways of water transport; and describe the role of essential nutrients.
enlighten ,describe and discuss about photosynthesis and its mechanism of action;
explain and discuss about mechanisms of enzyme action and respiration in plants;
discuss nitrogen fixation and various hormones and their action; and
appreciate how plants deal with stress and adapt themselves. 3 General Study Guide To get maximum benefit out of this study material please take note of the following points: i) Make a notebook with one side rule and the other side plain, keep a pen and some coloured pens/pencils with you. ii) Read the material slowly and attentively. Spend enough time on figures and flow charts. Try to draw the figures/flow charts and label them properly. This will help you in better understanding of the text. iii) While studying the text, underline the important points with a distinct colour (red, green, blue etc.) in the block itself. Write down salient points in the space provided on each page, or in your notebook, if necessary. iv) After finishing a section or subsection; ask yourself – what have I learnt? Try, to list the important points in you note book and compare them with the text and see if you have missed any. v) Attempt all the self-assessment questions (SAQs), wherever they appear. Don’t skip any of them as they are designed to assess your understanding of the subject. If you cannot answer, read the text again. vi) The answer to the SAQs and Terminal Questions are given at the end of each unit. Don’t get tempted to see the answers, before you try them. vii) If you don’t understand any word in the text, consult a dictionary. For scientific and technical words consult the glossary given at the end of each block or a scientific dictionary, if necessary. viii) For exploring the topics further, we have provided a list of suggested readings at the end of each block. 4  BBYCT-137 PLANT PHYSIOLOGY Indira Gandhi AND METABOLISM National Open University School of Sciences Block 1 WATER AND MINERAL NUTRITON UNIT 1 Plant-Water Relations 9 UNIT 2 Water Transport 33 UNIT 3 Mineral Nutrition 70 UNIT 4 Nutrient Transport 98 Course Design Committee Prof. G.C. Srivastava (Retd.) School of Sciences, IGNOU Former Head, Prof. M.S. Nathawat, Director, (Ex.) Department of Physiology, IARI, Pusa, Prof. Amrita Nigam New Delhi-110012 Prof. Jaswant Sokhi (Retd.) Prof. Vijay Paul Prof. Bano Saidullah (Retd.) Principal Scientist, Prof. Neera Kapoor Division of Plant Physiology IARI, Pusa, New Delhi-110012 Block Preparation Team Prof. Amrita Nigam Editor SOS, IGNOU, New Delhi-110068 Prof. G.C. Srivastava (Retd.) Dr. Eklavya Chauhan Former Head, Sr. Consultant, Department of Physiology, SOS, IGNOU, New Delhi-110068 IARI, Pusa, New Delhi-110012 Course Coordinator: Prof. Amrita Nigam Production Mr. Hemant Kumar SO(P), MPDD, IGNOU Acknowledgement Dr. Kumkum Chaturvedi for giving useful inputs. Sh. Manoj Kumar, Assistant for word processing and CRC preparation. March, 2021 Indira Gandhi National Open University, 2020 ISBN : All rights reserved. No part of this work may be reproduced in any form, by mimeograph or any other means, without permission in writing from Indira Gandhi National Open University. Further information on Indira Gandhi National Open University courses may be obtained from the University’s office at Maidan Garhi, New Delhi-110 068 or IGNOU website www.ignou.ac.in. Printed and published on behalf of Indira Gandhi National Open University, New Delhi by the Registrar, MPDD, IGNOU. Printed at  BLOCK 1 : WATER AND MINERAL NUTRITION Block I deals with the plant water relations, transport of water in a plant along with the nutrition of plants. Since plants have different modes of life than animals, there exist many differences in their physiology. This block primarily concerns with nutrition of plants, particularly the higher plants. A major difference between plants and animals, which influences the water relations of both, is the presence of cell wall in plants and its absence in animals. Plants need enormous amounts of water in order to live and grow. The unique properties of water make it an ideal elixir of life. The transport of water across the plant involves the existence of water potential and its components. Unit 1 and 2The water content and its availability and rate of involvement in soils depends upon the soil type and structure.The physicochemical properties of water make it an ideal medium for various biochemical reactions. Components of water potential operate for the uptake of water from soil to the specialised areas in the root like the root hairs. You will also study the movement of water through the apoplast, the symplast and the transmembrane pathway in the roots prior to its transport through the xylem conduits. In addition, the ascent of sap in a plant in governed by the forces created by the transpiration pull which helps water transport to great heights. You will also study different mechanisms involved in the opening and closing of stomata. Although minerals are transported along with water, the uptake of minerals into root cells involves special transport mechanisms, which are dealt in Units 3 and 4. In addition to gaining knowledge of essential elements in plants, you will also study about the criteria for essentiality, along with role of macro and micronutrients, and the techniques of studying mineral deficiencies. Also, the association between roots and the mycorrhizal fungi are vital for the uptake of certain elements like phosphorus, which are otherwise immobile. Unit 4 deals exclusively with the uptake of mineral nutrients into the roots via the Donnan free space and thereafter transport of ions across the cell membranes involving the membrane transport proteins, ionic pumps and ATPases.The radial movement of ions in roots as well as the mechanism of long distance transport involving xylem loading and unloading is also discussed in detail. Objectives After studying the block, you will be able to :
describe the importance of water to plants; enumerate the unique physico-chemical properties of water explain the various components of water potential and their relationship to osmosis and osmotic pressure;
describe the mechanism of absorption of water, and the different pathways of water transport; and different aspects of ascent of sap and transpiration; and
describe the role of essential nutrients; explain the uptake of nutrients by roots and the role of mycorrhizal associations. 7 Unit 1 Plant - Water Relation UNIT 1 PLANT –WATER RELATIONS Structure 1.1 Introduction Osmotic Pressure Objectives Diffusion Pressure Deficit (DPD) 1.2 Life Supporting Characteristics and Chemical Potential Biological Importance of Components of Water Water to Plants Potential 1.3 Water Transport Gradients of Water Potential Diffusion 1.4 Summary Imbibition 1.5 Terminal Questions Osmosis 1.6 Answers Membrane Permeability 1.1 INTRODUCTION Water is the most important single component which supports all living organisms on earth. All animals and plants, whether terrestrial or aquatic, are dependent on water for their survival. We could survive for some days without food, but it is impossible to live without water. Our daily weight fluctuates on account of excess or loss of water. Water is the most abundant component in any living system. It constitutes nearly 70% of the total body weight. In plant cells most of the water is localized in the vacuoles (upto 95%) and the rest resides in the peripheral cytoplasm. The amount of water in plant varies among different species depending upon their structure. Aquatic plants such as Chlamydomonas, Spirogyra, Chara, red algae, and others contain 97-98% of their weight as water. This is true also for Azolla, Eichhornia and other freshwater plants. However, among terrestrial plants, whether they are crops such as wheat, rice, maize, or trees such as sheesham, mango, neem and 9 Block 1 Water and Mineral Nutrition many others, the amount of water varies in different plant parts. It would be as high as 95% or more in young roots and as low as 30-40% in wood of a tree trunk. Young leaves often contain 85-90% water whereas mature leaves contain 60-80%. This means that the amount of water changes during the growth of the plant as well as during the growth of an organ such as leaf or seed. In this unit we will discuss the properties and importance of water for plants. Objectives Objectives After studying this unit, you should be able to:
explain the unique properties of water which make it the elixir of life;
differentiate between the processes of diffusion, osmosis and imbibition;
explain the various components of water potential and the relationship between them; and
describe and relate the movement of water in a single cell to a tissue and to the whole plant. 1.2 LIFE SUPPORTING CHARACTERISTICS AND BIOLOGICAL IMPORTANCE OF WATER TO PLANTS The life-supporting characteristics of water are due to its unique physical and chemical properties. Nearly 75% of our planet is covered with water in the form of oceans and other water bodies. The dominating presence of water in most cells must be seen in terms of its much larger number of molecules as compared to others. This is because water has a low molecular mass (18 Daltons). Water as a Chemical Reactant Water in a liquid state is an ideal medium facilitating the mobility of different macro/micro molecules within and across the cells. Water also influences the structure of different chemical components of cells. Water participates in many biochemical reactions either as a reactant itself (e.g., photosynthesis) or a product in others (e.g., respiration). During photosynthesis water splits in light reaction (photolysis) into oxygen and hydrogen. The latter is used in carbon fixation through CO2 for making glucose and constitutes the basic reaction of the autotrophic food chain. Various life characterizing biochemical reactions like condensation, hydrolysis, oxidation, and reduction occur in an aqueous medium. Water for Seed Germination Seeds do not germinate unless they get enough water to hydrate their protoplasm. Hydration triggers various metabolic reactions, and the 10 metabolites are mobilized in an aqueous medium. Unit 1 Plant - Water Relation Water as a Solvent Water is a polar molecule and has exceptionally high hydrogen bonding ability. This unique property allows it to dissolve a variety of molecules like sugars, amino acids, carrier proteins like hemoglobin and myoglobin. Sugars and proteins which contain polar –OH or –NH2 groups easily dissolve in water. Thus, an aqueous medium would be extremely/ highly suitable for uptake of dissolved mineral nutrients-so vital for growth and development of plants. It was precisely this property of water which made possible a variety of reactions in the “hot primeval soup” of the oceans during the origin of life. Thermal Properties of Water Water is unique as it remains in liquid form over a wide range of temperatures where life exists comfortably. If one considers the molecular size alone, water must be expected to exist in a gaseous state in most of the areas on earth. However, this is not true. The melting and boiling points of water are much higher than what would be theoretically expected. Presence of oxygen has introduced polarity and the tendency to form hydrogen bonds. It is this extensive hydrogen bonding which gives water its unique thermal capacity i.e., high specific heat, thermal conductivity, latent heat of vaporization and heat of fusion. Adhesion and Cohesion Adhesion and cohesion essentially refer to the “stickiness” that water molecules have for each other and for other substances. These attributes are due to the extensive hydrogen bonding. Water drop remains a drop on account of cohesion i.e., mutual attraction between molecules. For this to occur, the area of an air-water interface needs to be increased. This would require breaking of the hydrogen bonds and energy input. This energy needed to increase the surface area of a gas-liquid interface is called surface tension. High surface tension makes the water drops spherical and help movement of capillary water through the micro spaces. Another related characteristic is adhesion, which is the attraction of water to a solid phase. In plants,water “sticks” to the cell wall of the tracheary elements, i.e., xylem. This again is on account of extensive hydrogen bonding. Both adhesion and cohesion contribute to another related property, i.e., tensile strength. Tensile strength can be defined as maximum force per unit area that a continuous water column can withstand before breaking. This property is characteristic of metals. Water is an exception and exhibits a high tensile strength of 30 megapascals (MPa). The trio of adhesion, cohesion and surface tension gives water another unique property of capillarity, i.e., the ability of a water column to rise as a continuous column in plants. Providing Turgidity to the Cells Uptake of water results in turgor pressure which inflates the semi-elastic cellulosic cell wall resulting in initial volume expansion. Proteins, carbohydrates along with other metabolites are deposited only later during growth. 11 Block 1 Water and Mineral Nutrition SAQ 1 Match the terms in Column A with those of Column B Column A Column B a) Temperature regulation 1. Dipoles b) Mutual attraction 2. 0.239 calorie c) Hydrogen bond 3. Heat of vaporization d) Orientation towards (+ve) 4. 10 picoseconds and (– ve) ions e) Hydration 5. Germination f) Joule (J) 6. Cohesion 1.3 WATER TRANSPORT How does water flow in and out of the cells? How is water taken up from the soil by the roots? How does water escape from the plant into the atmosphere? To answer these questions, you would need to understand the dynamics of water flow with reference to the movement of substances and the different forces which are operative in this movement. Particles also exert forces by themselves. Before you study about them in detail, you must know that they are diffusion, imbibition, mass flow, and osmosis. Translocation is the term which broadly defines the movement of substances from one region to the other. The process of translocation may be passive on active, depending upon the need for expenditure of energy. The movement of water occurs through cell to cell by two principal pathways: a) Symplastic pathway where water moves from cell to cell via plasmodesmata. Since cells are joined via intercellular connections, the entire living portion of the plant forms a continuous single entity and is called symplasm. b) Apoplastic pathway is another route for the transport of water through cell walls, intercellular spaces, and non-living cells of the xylem. The non-living portion of plants is called apoplasm. 1.3.1 Diffusion You can smell from a distance the aroma of food being cooked in the kitchen. A bottle of perfume opened in one corner can be smelt in another corner. A crystal of copper sulfate dissolves in a beaker of water turning the latter blue. So does a drop of blue ink dropped in water. All the above phenomenon highlight a common point i.e., the random movement of particles is caused by 12 the inherent kinetic energy they possess. Unit 1 Plant - Water Relation The particles strike one another repeatedly thus, resulting in their dispersal in different directions. Also, these particles would always tend to move (perpetually move) in the direction where there is minimum obstruction offered by the other particles. It would mean, therefore, that the net movement would be favored from the area of higher concentration to the area of low concentration (region of higher free energy to a region of lower free energy). Thus, the spontaneous process or the tendency of particles (ions, atoms, or molecules) of gases, liquids and solids to get evenly distributed through the available space due to their random kinetic motion is called Diffusion (Latin diffuses = spread out). In other words, diffusion is a spontaneous process that leads to directed net movement of a substance from a region of higher concentration to a region of lower concentration, or simply as the net movement of molecules from a region of high free energy to a region of low free energy (see Fig 1.1). The term was coined by R. Brown in 1928. After sometime the particles/molecules will evenly spread throughout the available space. This would mean an equilibrium where the movement of particles will be equal in all directions, and thus net movement will be zero. The process of diffusion can be explained based on Fick’s First Law (a quantitative description of the process formulated by Adolf Fick in 1855), which in its oversimplified way suggests that the rate of diffusion is directly proportional to the cross-sectional area of the diffusion path and to the concentration vapor pressure gradient. At the same time rate of diffusion is inversely proportional to the length of the diffusion path. (a) (b) Fig.1.1: a) Diffusion of solute molecules from a region of high concentration (A) to a region of low concentration (B) till the molecules are uniformly distributed; b) At the time of uniform distribion, there is no net duffusion. It is believed that diffusion is most effective over shorter distances, as the average time required for a substance to diffuse for a given distance increases as the square of that distance. By this analogy, it is easy for glucose to diffuse in water, and with a diffusion coefficient of about 10-9 m2 s-1, a glucose molecule can diffuse across a cell of about 50/µm diameter in just 2.5 13 Block 1 Water and Mineral Nutrition seconds. Interestingly, the time taken by the same glucose molecule to diffuse to a distance of 1m in water will be nearly 32 years !!! Thus, diffusion is not much effective for translocation over long distances (see Unit 8). The ions particles/molecules which diffuse through the medium are the An example of solid to solid diffusion is diffusate while the medium into which these particles disperse is the when aluminum diffusant. For example, perfume is the diffusate and air is the diffusant; sugar diffuses into silver is diffusate and water is the diffusant. Diffusion can take place between gas- when both are placed gas, gas-liquid, liquid-liquid, solid-liquid and solid-gas and solid to solid in contact. phases. Rate of diffusion depends upon several factors. a) Temperature: Diffusion is directly proportional to temperature as it increases the kinetic energy of diffusate particles. Diffusion show a Q10 of 1.2-1.3. (The Q₁₀ denotes temperature coefficient, which is a measure of the rate of change of any biological or chemical system because of increasing the temperature by 10° C). b) Density of diffusate: Diffusion rate is inversely related to the square root of the density of the diffusate (d).  D∝ √ D = Diffusion rate, d = density of diffusate. Thus, gases diffuse more readily than do the liquids while the solids diffuse the slowest. c) The pressure exerted due to the tendency of the diffusate to diffuse is called its diffusion pressure.Because of their inherent kinetic energy, the movement of molecules will take place from the region of higher diffusion pressure to region of their lower diffusion pressure. d) Density and concentration of diffusant also determines/influences the rate of diffusion. More concentrated and dense the diffusant, lesser/slower will be the rate of diffusion. Importance of Diffusion The principle of diffusion is of great importance in plants as it is involved in the movement of almost all substances. Processes such as uptake of water and minerals, osmosis, transpiration, exchange of gases during photosynthesis and respiration, short distance translocation in the symplast, spreading of ions and other substances throughout the protoplast and wetting of cell walls, and release of odor for pollination depend in part on diffusion. 1.3.2 Imbibition Imbibition (Latin imbibere : to drink) is a process that involves absorption of water or any liquid by the solid particles of an absorbent substance without forming any solution. The liquid or water which is imbibed is called an 14 imbibate while the insoluble substance that imbibes water/substance is the Unit 1 Plant - Water Relation imbibant. The imbibants are usually in the form of colloidal particles which hold liquid water adsorbed on their surface as well as inside the minute spaces between the particles themselves. Although the liquid involved in imbibition in plants is water, other substance may also act as imbibants. For example, rubber imbibes ether/kerosene oil rather than water. Most important among the plant imbibants are the hydrophilic colloids like proteins, pectins, starches and cellulose. These substances differ in their imbibition capacities. The phycocolloid agar-agar has the maximum imbibition capacity and can imbibe almost 99 times its weight of water. Proteins also have a high imbibition capacity of imbibing nearly 15 times water their own volume. Cellulose and starch are poor imbibants and lignin does not imbibe water. Agar agar > oily seeds > proteinaceous seeds > starchy seeds. You must have observed at home that gram seeds when soaked, swell much more than those of rice or wheat. Since imbibition holds water in between the solid particles, the immobilized water loses its kinetic energy. The lost kinetic energy is released in the form of heat, often termed as heat of wetting. Kneading of wheat flour results in an increase in temperature. Moreover, imbibition results in increase in the volume (swelling) of the imbibant. However, this increase in volume is less than the volume of water imbibed. The process of imbibition develops a high pressure called imbibition pressure. It can be defined as the maximum pressure which can develop in an imbibant when brought in contact with water without allowing the imbibant to swell up. You will read in the following subsections that imbibition pressure is also referred to as matric potential. Dry seeds show a matric potential value of nearly –100 bars. Dry seeds like pea on encountering water can develop high imbibition pressure of up to 1000 atm (other equivalents used are bars and kg/cm2). 1 bar=0.987 atm=106 dynes/cm2=1.019 kg/cm2=14.5 lb./in2. This property has been exploited since ancient times to break rocks by filling dry wood in natural grooves of rocks and watering them. The wood swells up and cracks the rocks. Various factors influence imbibition: a) Rise in temperature enhances the rate of imbibitions. b) Texture of the imbibant, i.e., cohesion of molecules is inversely proportional to imbibition capacity. So, gelatin, with loosely packed particles will imbibe much more than hard wood. c) Since the imbibants are predominantly colloidal, imbibition is influenced directly by pH of the medium. For example, a negatively charged colloid like cellulose will imbibe maximum at alkaline pH. Since proteins are amphoteric, their absorption is maximum in other alkaline or acidic medium, but least in neutral medium. 15 Block 1 Water and Mineral Nutrition d) Electrolytes are imbibed at slower rates as they tend to naturalize the charges of the imbibants. e) Seed germination primarily depends on imbibition of water. f) Imbibition results in the breakage of seed coat. g) Many dehiscent fruits like pods of legumes, cotton bolls dehisce through imbibition in dry conditions by xerochasic, hygroscopic movements. Elaters of bryophytes also show hygroscopic movements. 1.3.3 Osmosis The term osmosis (Gr Osmos = impulse) was coined by Nollet in 1748 who first observed that a cylinder full of wine whose mouth was covered with animal bladder started swelling up and even burst when placed in water. Osmosis can be defined as the diffusion of water (or solvent) from a dilute solution into a stronger solution when separated by a semipermeable membrane. Thus, during the process of osmosis the direction and rate of flow depends on a semi permeable membrane, concentration difference and pressure difference in the two compartments. Osmosis can be demonstrated by a simple thistle funnel experiment which you must have performed in your schools. A solution which can cause entry of water into it is called an osmotically active solution. The osmotic entry of water into a system, organ or a cell from outside is called endosmosis, while exosmosis is the withdrawal of water from a cell or an osmotic system in response to a hypertonic solution. Swelling of water soaked dry raisins with intact stalks is an example of endosmosis while the fresh grapes shrink in size and become wrinkled due to exosmosis, when placed in 15% salt or sugar solution. Three types of solutions can be classified, based on the cell sap. a) Hypertonic solution : A solution having more concentration than that of the cell sap. If a cell is placed in such a hypertonic solution, water will diffuse out due to exosmosis resulting in shrinkage of the protoplasm (plasmolysis). b) Hypotonic solution : In this case, the concentration of the external solution is less than that of the cell sap. When a cell is immersed in a hypotonic solution, water diffuses into the cell (endosmosis) resulting in an increase in cell volume. c) Isotonic solution : When the cell sap and the external solution have the same concentration. There is no change in the cell volume as there is no net movement. 1.3.4 Membrane Permeability The tendency or ability of a membrane to allow or restrict the passage of substances through it is referred to as Membrane Permeability. This is a 16 characteristic feature of all membranes. Rate at which the passage of solute Unit 1 Plant - Water Relation particles is allowed through the membranes varies with the permeability property of membranes. For example, a permeable membrane allows the passage of both the solute as well as the solvent particles through it. Majority of cellulosic cell walls behave as permeable membranes. On the other hand, the suberized walls of cork cells, or cutinized walls do not allow the passage of any substance and are, therefore, impermeable. Semipermeable are those membranes which allow only the solvent but not the solute particles to pass through e.g., parchment, copper ferrocyanide membrane or collodion membrane. Plasma membrane, tonoplast and membranes of organelles have unique permeability characteristics. These differentially permeable or selectively permeable membranes allow the passage of solvent (water) as well as selected (specific) solutes by a variety of mechanisms, operative within their lipoprotein complexes. For example, special protein-lined channels called aquaporins are responsible for water absorption and its further packaging. In addition, ions of solutes are transported through special ion channels (approximately 30 kDa). 1.3.5 Osmotic Pressure It is the hydrostatic pressure developed in a solution which is just necessary to prevent the flow of water or a pure solvent to a solution (osmosis) when the two are separated by a semi-permeable membrane. In other words, it is the maximum pressure which can develop by an osmotically active solution when it is separated from its pure solvent by a semi permeable membrane. Osmotic pressure (OP) can be measured by an osmometer, which involves a manometer. Pfeffer’s osmometer and Hartley’s osmometer are the two most used instruments for this purpose. The former employs very dilute solutions whereas the latter set up uses an external pressure source to counter the osmotic pressure. Since both the entities are equal, OP can also be alternately defined as the pressure that is required to be applied to a solution in order to prevent an increase in its volume due to the inherent tendency of water (on solvent) to enter it, when the two are separated by a semi-permeable membrane. The value of OP is directly proportional tothe concentration of the solution. The highest value of OP is observed in halophytes (>200 atm). Osmotic pressure is denoted by the following equation given by van‘t Hoff (1887). OP = m RT or OP = c RT Where OP = osmotic pressure m/c = molar concentration of solution (concentration of solute/litre) R = Universal gas constant (0.082 atm/mol) T = Absolute temperature in Kelvin (K) = (t + 273) t = temperature in °C Theoretically, OP of one mole glucose solution at 0°C will be OP = 1 × 0.082 × 273 17 Block 1 Water and Mineral Nutrition 22.38 = # 22.4 atm, = 22.69 bars (1) = 2.269 MPa = 22.69 ×105 Pa, and at 20°C will be = 1 × 0.082 × (273+20) = 1 × 0.082 × 293 (2) = 24 atm = 24.31 bars = 2.431 MPa = 24.31 ×105 Pa The above-mentioned formula for OP is applicable to non-electrolytes e.g., glucose and sucrose. For electrolytes like NaCl or KCl, the degree of dissociation will also have to be considered. For electrolytes, the formula becomes OP = ciRT Where i is the van ‘t Hoff’s ionization constant. Ionization increases the number of particles in a solution. Thus, it is natural that the same volume, of electrolyte would exert more OP than its equimolar non-electrolyte counterpart at the same temperature and pressure. To give you an example, 0.01 M glucose (non-electrolyte) will have an OP of 0.24 atm whereas equimolar sodium chloride (electrolyte) will have an OP of about 0.47 atm. Thus, the OP of an electrolyte is greater than that of the equimolar non- electrolyte. Therefore, water moves from lower OP towards the higher OP. Turgor Pressure (Hydrostatic Pressure/Pressure Potential) The pressure developed by the turgidity in a cell is called turgor pressure (TP). Turgor pressure is applicable to living cell and not to a free solution. TP is called as hydrostatic pressure or pressure potential. When a cell is immersed in water, water enters the cell as cell sap has a higher osmotic pressure. It causes swelling of the osmotic system. As a result, the hydrated cytoplasm presses against the cell wall. The outward force applied by the turgid protoplast on the cell wall is called turgor pressure (Fig.1.2). Since the cell wall is semi-elastic, it expands to a certain extent. The cell wall prevents a plant cell from bursting, when placed in water (hypertonic solution). An animal cell in a similar situation will burst. The cell wall exerts an equal and opposite pressure to counter the turgor pressure and it is called the wall pressure (WP). Thus, the TP and WP will be equal but exerted in opposite directions Fig. 1.2). TP = WP Turgor pressure keeps the protoplast hydrated and appressed to the cell wall. A fully (turgid) hydrated cell will have maximum hydrostatic pressure which will be equal to its OP. On the other hand, TP = 0 in a flaccid cell. The value of TP would, therefore, lie between zero and the value of OP. Under exceptional 18 conditions of plasmolysis, TP may even become negative. Unit 1 Plant - Water Relation Fig.1.2: Osmotic relations exhibited by a plant cell. Turgor pressure helps in keeping the protoplasm in a hydrated state which is important to maintain the 3-D structure of the organelles for their proper functioning. In addition, turgor pressure helps in cell elongation, germination and brings about a variety of plant movements including the seismonastic movements as well as opening and closing of stomata. 1.3.6 Diffusion Pressure Deficit (DPD) Each liquid possesses a definite diffusion pressure with which it can move. Seismonastic Pure water has maximum diffusion pressure. Addition of solute to it reduces its movements are diffusion pressure. So, the diffusion pressure of a solution is always less than paratonic movements its pure solvent. This reduction in the diffusion pressure of water in a solution brought about by or system over its pure state due to presence of solutes is called Diffusion external mechanical Pressure Deficit (DPD). In other words, DPD is the difference between the stimuli like vibrations, diffusion pressure of a solution and its pure solvent when present at the same strong winds, rain drops, or contact with temperature and pressure. The term DPD was coined by Meyer (1938). This a foreign body. term is preferred over the earlier term Suction Pressure (SP) proposed by Renner (1915). DPD constitutes the power of absorption by the cell and determines the direction of osmosis. The earlier term SP was used to denote the amount of pressure by which water can be sucked into the cell or likewise, expelled out of the cell. Another way to define DPD will be that it is the amount of water needed by a cell to make it fully turgid, as it is a measure of the water absorbing capacity of a cell. A solution will absorb more water or solvent molecules to equalize its deficit of diffusion pressure. So, water will always move from low DPD to high DPD. Water Movement Lower DPD Higher DPD The value of DPD is always positive for a cell and is equal to osmotic pressure of the system minus wall pressure. DPD (SP) = OP – WP (=TP) When a cell is placed in pure water or in a less concentrated solution (hypotonic) than that of the cell sap, water enters the cell, resulting in an 19 Block 1 Water and Mineral Nutrition increase in TP. Simultaneously the OP of the cell goes on decreasing as there is a continuous inflow of water and the concentration of the cell sap goes down. The value of TP goes on increasing and eventually becomes equal to OP. At this stage, the cell becomes fully turgid DPD = OP-TP when OP = TP; then OP-TP = 0 DPD = 0. Thus, a fully turgid cell does not have any capacity to suck water further. On the other hand, when a cell is placed in a hypertonic solution, water from the cell goes out by the process of exosmosis. As a result, the OP increases and these in a corresponding decrease in TP. The cytoplasm contracts and eventually the protoplast leaves the cell wall. This flaccid cell is said to be plasmolyzed. At the time of complete plasmolysis, value of TP becomes zero. So, at complete flaccidity, since TP = 0 and DPD (SP) = OP-TP DPD = OP Such a cell will have maximum absorptive capacity. If a cell was placed in some hypotonic solution other than water, then its DPD (SP) = (OP-OP1) – TP (OP1, is osmotic pressure of the hypotonic solution) Sometimes the value of TP becomes negative when the cell wall of the plasmolyzed cell pulls water in the opposite direction Since TP is –ve DPD = OP – (-TP) DPD = OP + TP Thus, the DPD of the plasmolyzed cell is greater than the value of OP. It is clear from the above that the DPD of plant cell is not directly proportional to osmotic concentration of the cell. It depends on OP as well as TP of the osmotic system. So, it is DPD which is the basic determining force with which a cell or an osmotic system will lose or absorb water, rather than the OP and TP individually. SAQ 2 In the following statements fill in the blank spaces with appropriate words: a) Cells undergo plasmolysis when kept in ……………….solution. b) Osmosis is diffusion of water through ………………. from a region of high concentration of water to a low concentration of water. c) In a hydrated cell, the hydrostatic pressure will be ………………, and equal to its ……………… 20 Unit 1 Plant - Water Relation 1.3.7 Chemical Potential The chemical potential of a substance in a system is a measure of its free energy. The free energy denotes the potential for performing useful work or the energy available to do work, which is force x distance. Here the system refers to the thermodynamic concept wherein studies are of system rather than individuals or bodies. Thus, when we study the properties of a solution in a container, we are studying a system wherein each individual component interacts internally. For example, suppose we have pure water in a system, be it in a beaker or soil, all the molecules of water can do work, so their free energy is maximum. When a small amount of sugar is added a few molecules of water get associated with each molecule of sugar, the free energy of water decreases. Hence, the free energy of pure water is maximum, and addition of any solute lowers the free energy or chemical potential of water. This shows that the chemical potential is a relative quantity, which represents the difference between the free energies (potential) of the substance in each state to that substance in a standard state. The unit of chemical potential is energy per mole of substance (J mol-1). The chemical potential of water in a solution is related to is vapour pressure and is given by the equation  μ −μ ° =   °  ∆ =   ° Where: µw = chemical potential of water in question (joules/mole) µw° = chemical potential of pure water under STP ∆µ= change in free energy R = gas constant T = Absolute temperature in Kelvin e = vapor pressure of water in question Note that  Relative Humidity = ° × 100  If it is pure water, then  = 0 and ∆µ also becomes 0. ° Partial Molal Volume So, the chemical potential of water is set to zero. If chemical potential is less  (J) of a solution is the than that of pure water then  ° will be negative number, and therefore, ∆µ change in volume of a will be less than zero, a negative number. So, the maximum value of chemical solution when one potential is 0, and addition of the solute lowers it to negative values. Water will mole of a substance spontaneously flow from regions of higher chemical potential to those is added to it. possessing lower water potential. 21 Block 1 Water and Mineral Nutrition 1.3.8 Components of Water Potential A related term Water potential (Stalyer and Taylor, 1960) has been used Water potential is historically by the plant physiologists, which is the difference between expressed in pressure units. Energy per unit chemical potential of water at any point in a system and that of pure water at volume of water is STP (standard temperature and pressure). By conversion, the chemical expressed in joules potential of water is referred to as water potential. It is denoted by the Greek 2 per cm. 106 dynes=1 letter Ψ (-pronounced as psi). bar. The present unit used for pressure is Ψ w is expressed in pressure units. It is the free energy per unit volume of pascal (Pa). It is a water (Jcm-3). Ψw is equivalent to the chemical potential of water (∆µ) divided pressure equal to the by the partial molal volume of water , i.e., the volume of 1 mol of water, which force of one Newton is 18 × 10-6 m3 mol-1). acting uniformly over one square metre. If water potential of the source (the region supplying the water) in higher than 5 the water potential of the sink (the receiving region), then there is a 1 bar = 10 Pa spontaneous transfer of water from the source to sink (Ψsource > Ψsink). 3 10 Pa= 1 KPa (1 kilo pascal) By convention, the water potential of pure water at atmospheric pressure is 3 taken as zero. Therefore, water potentials other than that of pure water will 10 KPa = 1MPa (mega pascal) generally be negative. Thus, lower potential means a more negative value, and higher potential is less negative value. Since the water potential of a system is related to the difference in the vapor pressures of water in a solution and pure water state, it is taken as equivalent to the diffusion pressure deficit (DPD). As explained earlier, Ψw of pure water is 0, and so is the value of DPD. Addition of solute (now a solution) will have a water potential as negative, whereas the DPD would increase (greater than 0). You will read in the next sections that the direction of movement of water is from lower DPD to higher DPD. On the other hand, water moves from a region higher (less negative) water potential to lower (more negative) water potential. Water potential in plants is affected by three major factors: solute concentration, hydrostatic pressure, and matric forces. In addition, gravity also plays some role. Water potential (Ψ/Ψw) is equal to the algebric sum of the potentials – Solute potential (Ψs), pressure potential (Ψp), matric potential (Ψm) and gravitational component (Ψg – when present). Ψw = Ψs +Ψp + Ψm + Ψg Let us consider each of these components in details: Effect of Solute Let us take pure water in two chambers A and B separated by a semi- permeable membrane (Fig. 1.3a). The water potential of both the chamber is zero. Addition of solute in chamber A (Fig. 1.3b) would reduce the free energy of water and the water potential will fall below zero. Consequently, water will move from B to A (Fig. 1.3c). The effect of dissolved solutes on water potential (Ψw) is called osmotic potential (Ψπ). It can be estimated numerically if we know the osmotic pressure of the solution. The two are related as π = –Ψπ 22 For example, if π of solution is 5 bars then Ψπ would be –5 bars. Unit 1 Plant - Water Relation Osmolality = moles of total dissolved solutes per litre of -1 water – (mol L ). 6.02 23 × 10 particles of the non-ionic solute will be present in one litre of solution with -22.7 bars at O°C = 1 mole of non-electrolyte and -48.3 bars for the electrolyte sodium chloride. Fig.1.3: Experiment to show the effect of solute and pressure on water potential. In other words, osmotic potential or solute potential (Ψs) represents impact of dissolved solutes on its water potential i.e.it records the decrease in the water potential or free energy of water due to the presence of solute particles (ionic or non-ionic). Under ideal conditions, the Ψs is also equivalent to the maximum osmotic pressure which can be developed under ideal conditions but written with a negative sign as the dissolved solutes reduce the Ψw of a solution with reference. Dilute non-ionic solutions, e.g., sucrose and glucose will have an osmotic potential (solute potential). Ψs = –RTCs (see osmotic pressure Section 1.3.5 for values of R and T) Osmotic potential of 1.0 molal solution of sorbitol at 20°C will be [Ψs = –ci RT] : –(1.0mol kg-1) (1.0) (0.00831 kg MPa mol-1 k-1/0.080205 kg atm mol-1 k-1) (20 + 273 = 293°K)= 2.43 MPa] The solute potential of the electrolyte will decrease by the degree of its dissociation over that of a non-electrolyte. To be expressed as Ψs (electrolyte) = iRT. By this analogy, 0.33 M calcium chloride solution will have the same solute potential as 1.0 M sucrose. Effect of Pressure Let us now see the effect of pressure on water potential. As Fig. 1.3d illustrates, when pressure is applied the flow of water begins from chamber A 23 Block 1 Water and Mineral Nutrition to chamber B through a semi-permeable membrane. This means that pressure increases the free energy of water and thus raises the potential of pure water above zero. The effect of pressure on water potential is called pressure potential and is designated as Ψp. The level of water in B rises due to increase in water potential of A (Fig. 1.3 e). Pressure potential is usually positive (is equal to turgor pressure). The values of turgor pressure are particularly high in leaf cells of crop plants during warm afternoons and in guard cells during stomatal opening. In sharp contrast, xylem and in the walls between cells, a negative hydrostatic pressure or tension develops, leading to the value of Ψp becoming negative. Now see the Fig. 1.3 where two chambers are present. What would happen if pressure were applied on the chamber containing solutes (Fig.1.3 d)? Now the Ψw will be affected by both solute and pressure. The solute would lower the water potential and pressure will raise the water potential. So, the flow of water from B to A would start decreasing (Fig.1.3 e). An equilibrium condition will reach when pressure potential Ψp will be equal but opposite in magnitude to osmotic potential Ψπ and there will no net flow of water in the two chambers (Fig.1.3 f). This can be represented by the following equation: Ψw(B) = (Ψπ + Ψp)B …(1.1) Let us suppose if Ψp is equal and opposite to Ψπ. Then ΨWA = (ΨπA + ΨπB) =0 We are going to discuss another important factor, i.e. the “Matric pressure”. Effect of Matric Pressure Water can get absorbed to the wettable surface of solids such as soil, wood, seeds, and cellular constituents. A force operates between solid-liquid interface and is called matric suction or matric pressure. The absorption process is accompanied by heat loss and results in decrease in free energy of water. In other words, the effect of matric forces on water potential is called matric potential (Ψm). Its value is always negative. Matric potential can be viewed as a decrease in water potential on account of immobilization of water molecules due to their adsorption over the colloidal particles in the cytoplasm and cell wall. The value of Ψm may be appreciable in young cells, dry seeds, fruits, and some extreme xerophytes. The mature cells of mesophytes, however, show an extremely low Ψm value of –0.1 bars, and thus, can be ignored. Likewise, in a well-watered soil Ψm is not very significant, however, when the soil is nearly drying Ψm determines water potential of the soil. It is very natural that the water column will be forced to move downward due to the gravitational force. Thus height, density of water and the acceleration due to gravity will play a role to decide the equal and opposite force to counter this gravitational potential– Ψg. It has been calculated that the value of Ψg is quite insignificant (accounting for nearly 0.1 MPa in water potential) and is, 24 therefore, generally not considered for water transport at the cell level. Unit 1 Plant - Water Relation So, we find that the Ψm is composed of the following main component forces: Ψw= Ψπ+Ψp + Ψm+ Ψ … (1.2) Ψ= any other forces that may influence Ψw. Thus, water potential is equal to Ψw= Ψs= Ψp Resistances to Water Movement and Water Flux We have explained earlier that if water potential drops from source to sink st Fick’s 1 Law : (Ψsource>Ψsink) then there will be spontaneous flow of water. But we do not Diffusive flux = know the rate of this transfer i.e., flux. Let us learn how to calculate flux. diffusion coefficient; concentration Flux describes any effect that appears to pass or travel (whether it moves or gradient not) through a surface or substance. With reference to the transport J = Ds∆/∆x phenomena, flux is considered as a vector quantity (having both magnitude J = Flux rate and direction) which describes the magnitude and direction of the flow of a 2 (moles/m ) substance. The diffusive flux can be related to the concentration gradient using Fick's first law, which postulates that the flux moves from regions of ∆Cs/∆x is concentration high concentration to regions of low concentration. The magnitude of flux is gradient proportional to the concentration gradient. In other words, the solute moves -3 (moles m /m) from a region of high concentration to a region of low concentration across a Ds = diffusion concentration gradient. 2 -1 coefficient (m s ) The flux for water flow is denoted by Jw which is volume of water flow through unit surface area per unit time. Water in plants flows from cell to cell and, also through cell walls. When water moves from cell to cell the flow is the function of water permeability of the membrane. The flux of water is given by Jw = Lp ∆Ψw … (1.3) Lp = permeability coefficient of limiting membranes ∆Ψw = difference in water potential at two points From equation (1.1) we know that Ψw = (Ψπ + Ψp) ∆Ψw = (∆Ψπ + ∆Ψp) substituting the value of ∆Ψw in above equation (1.3) Jw = Lp(∆Ψπ + ∆Ψp) …(1.4) Thus, the inward or outward rate of flow or water from cell to cell and tissues can be calculated from the above expression. The water flux is directly proportional to ∆Ψ and inversely proportional to hydraulic resistance (R). In other words, higher the ∆Ψw ,more will be flux, but high R will decrease the flux. In plants water will move through the pathway which offers least resistance. Between the two routes – cell walls and cell to cell, the membranes of the cells exert more resistance (because of low permeability) than the cell walls. 25 Block 1 Water and Mineral Nutrition Therefore, water can flow relatively easily through cell walls. Water will not Hydrostatic experience the resistance of plasma membrane when it moves from cell to cell pressure is the pressure exerted by via plasmodesmata. The xylem conduits which are not obstructed by cell or on a liquid above membranes have least resistance and the rate of flow of water is very high. or below atmospheric The ratio of resistance in xylem, cell walls and cell membranes is in the order pressure. of 0.3:1: 50. This explains why xylem is the pathway for long distance transport as has been observed experimentally. The resistance in xylem varies inversely with the diameter of xylem elements. The smaller the diameter of xylem greater will be the resistance. In soil, pressure potential is insignificant and osmotic potential is zero because there are no membranes (solute and water move together). Hence, the driving force ∆Ψw in soil is determined by the matric pressure. ∆Ψw(soil) = – ∆Ψm(soil) SAQ 3 a) List the three factors that determine the value of Ψw in plant. b) The water potential in a cup (Ψc) containing salt solution will be Ψc>Ψw Ψc H+> Ca2+>Mg2+> K+ = NH4+> Na+ which indicates that the in order of their ability monovalent cations will be less strongly retained than the divalent ones and to salt out or salt in the trivalent ions will have the highest binding affinity. Since cation adsorption proteins. First worked is a reversible phenomenon and any ions with a lower affinity (say Ca2+) can out by Franz be replaced with that a higher affinity (say H+), this ion exchange capacity or Hofmeister, the series ease of removal follows a pattern reverse of the lyotropic series. On that is also called as Hofmeister series. account, sodium ions are exchanged most readily while aluminium ions show The series are least exchangeability. arranged in an ascending or The colloidal fraction with its adsorbed ions represents the main nutrient descending order of reservoir. However, it is the ions that are present in the soil solution which their influence on the constitute the immediate source of mineral nutrients to the plants. We can properties of the view the entire soil system as a dynamic one where weathering of rocks solvent and on the particles are releasing ions that continuously replenish the colloidal reservoir. degree of the These exchangeable ions held by the colloidal system replace/replenish the chemical reactions and the free ions in the soil solution which have been taken up/consumed by the roots. physicochemical A careful study of the above Table 4.1shows the following: processes occurring there. i) The concentration of potassium, phosphate and nitrate declined significantly in the bathing medium within four days. ii) The concentration of sodium and sulphate, however, increased indicating that water was absorbed faster than either of the two ions, or possibly they leach out. iii) The rate of uptake especially of potassium and calcium, differed between the two plant species, maize, and bean. iv) The ion concentration (particularly of K + NO 3− and SO 24 − ) in the root was 100 considerably higher than in the nutrient solution used for the experiment. Unit 4 Nutrient Transport These results show certain characteristics of nutrient uptake. 1. Selectivity: Certain mineral elements are taken up preferentially while the others are discriminated against or nearly excluded. 2. Accumulation: The concentration of mineral elements can be much higher in the plant sap than in external solution. This means the uptake is against concentration gradient. 3. Genotype: The nutrient uptake capacity of various plant species differs due to their different genetic makeup. You know that chemically Na+ resembles K+ very closely but the rate of absorption of K+is not influenced by similar concentration of Na+ ions in the medium. The process of K+ absorption is, therefore, selective, and uninfluenced by a related ion. Similarly, several other monovalent and less related divalent ions also have no effect on K+ uptake. Likewise, absorption of - Cl is unaffected by related halides, fluoride and iodide, as well as other anions like NO 3− , H 2 PO 24 − or SO 24 −. However, interestingly, Ca2+ is an absolute requirement for this selectivity. For example, in the absence of Ca2+, the absorption of K+ is inhibited by Na+. Even though the ion uptake mechanism is highly specific, yet it can often ‘fooled’ by similar ions. For example, it has been seen that absorption of K+ can be competitively inhibited by rubidium ( Rb+ ). Similarly, competitive 2+ 2+ inhibition of Cl − by Br , Ca , Sr 2+ by Mg , and sulphate by selenate − 2− ( SeO 4 ) has also been observed. The selectivity and the rate of uptake of the nutrients and metabolites are influenced by temperature, O2, poisons, carbohydrate content of the tissues and light. Such effects are like enzyme-mediated reactions and indicate that proteins are involved in solute uptake. You will study about transport proteins and mechanism of ion uptake later in this unit. 4.2.3 Movement of Nutrients in the Root In the previous Units1 and 2, we discussed about the two main routes – apoplast and symplast – by which water and dissolved solutes are conducted across the root interior into xylem vessels and tracheids. The cell wall spaces and intercellular spaces in the root’s epidermis and cortex are virtually continuous with the external soil solution. As was mentioned in Unit 2, ions can move up into the root hair as well as epidermal cells. An ion that is absorbed by an epidermal cell and moving towards the xylem in the symplast must cross the epidermis, several cortical cells, the endodermis and the pericycle. The movement would involve transport directly through each of the two primary walls, middle lamella, and plasma membranes between the cytosol of adjacent cells. Alternatively, the solute can move through plasmodesmata without crossing the plasma membrane or diffusing through the cell walls. The nutrient movement along the apoplasm is prevented at root endodermal cells because these cells are lined with Casparian strips. Therefore, the water 101 Block 1 Water and Mineral Nutrition and dissolved substances must enter the cell and pass through them via the symplastic route. Thus, all minerals must pass through the cytoplasm to reach the xylem. With this background we are now ready to trace the path of a solute (or nutrient) entering the root from the surrounding soil solution. If the cell full of ions is returned to a lower-salt solution, some of the absorbed ions will diffuse out into the external medium. This process of free diffusion of solutes follows the simple laws of diffusion. This free diffusion means free movement of ions in and out of the tissue. This part of the cell wall tissue is referred to as Outer Space. 4.2.4 Free Space The first step in mineral uptake is the diffusion of ions through the cell wall into the apparent free space. You have already studied that the primary cell wall of the plant cell consists of mainly cellulose microfibrils which are embedded in an amorphous matrix of two polysaccharides, hemicelluloses and pectic substances. Hemicelluloses are made of sugars other than glucose (e.g. xyloglucans) while pectic substances are partly made from polygalacturonic acids. These acids have weak carboxylic acid groups (-COOH) that ionize and give rise to negative charges ( − COO − ) on which hydrogen ions are loosely held. When positively charged ions such as K+, Mg+2, Ca2+ pass through the plant cell wall they displace hydrogen ions of the carboxyl groups and are held there by the weak inter-ionic attractive forces. The negative charges of the COO- group cell wall are termed cation adsorption sites or cation exchange sites or Donnan Free Space. A cation such as Ca2+ with a relatively high adsorptive capacity will displace ions with a lesser adsorptive affinity (e.g. K+). The cellulose microfibrils are not very tightly packed; as a result, they leave small pores between them. The pores are large enough to allow free movement of water and dissolved substances. The diameter of these pores is in the range of 5.0 nm whereas, the dimensions of hydrated ions such as K+ and Ca2+ are smaller (Table 4.2). So, the pores do not restrict the movement of ions. Water and dissolved nutrient molecules, ions, and metabolites of the size of glucose, sucrose, and amino acids diffuse readily across primary cell walls. Table 4.2: Relative sizes of pores and molecules Comparative Cell Wall of Maize 100-200 Pores of Cell Wall > 5.0 Sucrose 1.0 Hydrated ions + K 0.66 2+ Ca 0.82 The intercellular spaces, the negatively charged regions (Donnan Free Space) in the amorphous matrix and the pores in the cellulose microfibrils are readily 102 accessible to water and dissolved ions. The fraction of the volume of the plant  Unit 4 Nutrient Transport tissue readily accessible to diffusion of an external solute dissolved in water is termed ‘Free space’. The free space in the root is bound by the plasma membrane of the epidermal and cortical cells and the casparian strip of the endodermis. Any substance which can easily pass through the free space reaches the external surface of the protoplasm. Here, it encounters the plasma membrane which is an effective barrier to its movement further inward. Nevertheless, the plasma membrane does not act like a passive barrier, it can allow the passage of some substances into the cell interior while selectively inhibiting the passage of certain others. In the following section, we will discuss the transport of nutrients across the plasma membrane. Apparent free space (AFS) is not separated from the environment by any membrane and ions can enter it by free diffusion. This space is negatively charged like the colloids and can also hold some cations by electrostatic attraction. Thus, this space denotes that portion which accommodates ions by free diffusion and where some ions are held electrostatically. The AFS is estimated around 10-25 percent of the root volume. The AFS consists of cell walls and the intercellular spaces, areas considered to be equivalent to the apoplast of the root epidermal and cortical cells which the ions can enter without crossing the membranes. SAQ 1 a) State whether the following statements are true/false. i) The apparent free space (AFS) is negatively charged. [ ] ii) The colloidal particles of soil provide a small surface area for interaction of mineral elements. [ ] iii) The exchange capacity of mineral ions is independent of pH of soil. [ ] iv) Passive diffusion is unaffected by use of metabolic inhibitors. [ ] v) Sodium ions are the most readily exchanged cations. [ ] b) Tick mark the correct alternative from the words given in each parenthesis. i) The clay micelles have (negative/positive) charge. [ ] ii) Cytoplasmic strands connecting the adjacent cells are (plasmodesmata/casparian strip). [ ] iii) The ion exchange capacity is affected by (temperature/pH) of the soil. [ ] iv) The apoplastic pathway is broken at the (casparian strip/ plasmodesmata) of the endodermal cells. [ ] v) The fixed negative charges formed by the weakly acidic carboxyl group of polygalacturonic acids form (cation exchange sites/anion exchange sites). [ ] vi) K+ absorption can be competitively inhibited by (sodium/ rubidium). [ ] 103 Block 1 Water and Mineral Nutrition 4.3 TRANSPORT OF IONS ACROSS PLASMA MEMBRANE After entering the free space, the mineral nutrients must now cross the plasma membranes before entering the root and begin long distance transport to the shoot. Transport across the root membranes follows certain rules based on our current knowledge of membrane composition, architecture, and function. Uptake of ions may be passive (occurring due to physical forces, independent of temperature, and unaffected by the application of metabolic inhibitors) or active (possible with expenditure of metabolic energy). Passive uptake of ions may also be mediated by transport proteins which facilitate the passage of selected ions and molecules. This includes three main types, viz., channels, carriers, and pumps. Primary active transport requires ATP to undertake a transport against the concentration gradient with the help of electrogenic pumps. Secondary active transport, on the other hand uses stored free energy (in the form of H+ gradient) and indirectly driven by pumps like the proton motive force. In this section, we will study the transport of solutes, particularly inorganic ions across the plasma membrane, their entry into the xylem elements and long- distance transport from root to shoot. The transport of substances across the membrane follow three basic modes, viz., simple diffusion, facilitated diffusion, and active transport (diagrammatically depicted in Fig. 4.4). Fig. 4.4: Different routes for transportation of molecules across the plasma membrane (From Taiz et al). 4.3.1 Simple Diffusion Simple diffusion is one of the means for transport of ions across the plant cell membranes (Fig. 4.5). An artificial or synthetic lipid membrane made in the laboratory allows the non-polar solute molecules to pass through it more readily than the polar molecules. Over a period, these molecules can diffuse across the lipid bilayer down its concentration gradient by virtue of their own kinetic energy. However, the rate at which a molecule diffuses across a lipid bilayer depends on the size of the molecule and its relative solubility in the lipid. Non-ionic hydrophilic substances are generally taken up as inverse function of their size, whereas hydrophobic substances are transported as a 104 function of their lipid solubility. Unit 4 Nutrient Transport Fig. 4.5: The relative permeability of an artificial lipid bilayer to different class of molecules. Small non-polar and hydrophobic molecules like O2 and N2 readily dissolve in lipids and therefore, rapidly diffuse across the bilayer. Ethanol (46 Da), carbon dioxide (44 Da) and Urea (60 Da) cross the bilayer rapidly while the larger glucose molecules (180 Da) do not. Interestingly, the water molecules (18 Da), even though polar and relatively insoluble in lipids diffuse very rapidly across the lipid bilayer through special water-selective channels or aquaporins. Membrane lipid bilayers are relatively impermeable to charged ions, despite their small size. This is because the charge and large hydration shell around them prevents them from entering the hydrocarbon phase of the bilayer. Lipid bilayers are 109 times more permeable to water than to small ions as Na+ or K+ (Fig. 4.6). K+ is more permeable than most ions whose permeability coefficient is arbitrarily set at 1.0. It is taken as a standard in solution to which the permeability of their ions is compared. However, some solutes like O2, CO2 and NH3 are non-polar and readily diffuse through the lipid bilayer of the membrane. The ability of lipid bilayer to discriminate between low and high molecular weight hydrophilic materials, permitting the former to diffuse across but not the latter is due to the presence of pores in the bilayers. These pores, called “statistical pores” are formed randomly because of thermal movement of acyl phospholipid chains. Since these pores are only transient, they cannot be viewed even under the transmission electron microscope. Transport by diffusion is a passive process and does not require a direct input of ATP. The driving force for diffusion comes from the concentration or electrochemical gradient of the solute being transported and obeys Fick’s Law, which states that the rate at which molecules in a solution diffuse from one region to another is a function of their concentration difference. In other words, the rate of movement of a substance is directly proportional to the concentration gradient. As a result, solutes do not get accumulated against this gradient. 105 Block 1 Water and Mineral Nutrition -1 Fig. 4.6: Permeability coefficients cm s for the passage of various molecules through artificial lipid bilayers. 4.3.2 MembraneTransport Proteins Despite having some similarities, biological membranes differ on many counts with the artificial membranes. It has been recognized since long that certain ions enter the cells much rapidly than the lipid bilayer would theoretically allow them to do so. Even though the lipid bilayers do not permit the entry of polar molecules such as ions, sugars, amino acids, nucleotides and cell metabolites, these substances do pass through the biological membranes. For this purpose, the biological membranes contain number of proteins, called transport proteins. Many of these specialized proteins facilitate or mediate diffusion of ions or charges solutes, which is otherwise not possible through a pure lipid bilayer. The solubility problem is overcome and this rapid, assisted diffusion of solutes across the membranes has been termed facilitated diffusion. Since the concentration gradient (for uncharged solutes) or the electrochemical gradient (for charged solutes and ions) determines the direction of transport, i.e., it operates along a concentration gradient, it is also called passive-mediated transport. There are three classes of membrane transport proteins, viz., channels (pores-simple and gated), carriers (transporters) and pumps (for pumping solutes against their concentration gradient or electrochemical potential by 106 directly using ATP). Unit 4 Nutrient Transport Channels We can visualize channel proteins as charged-lined, water-filled channels of transmembrane proteins which extend across the membrane and act as selective pores. They allow specific ions or molecules to permeate through the channels. The transport specificity of these channels is determined by the size of the pore, the nature and density of the surface changes present on their inner lining. The channels are named/ identified based on the type and the hydrated size of ion species they allow. Only passive transport takes place through these channels and by virtue of the electric charge and pore size only water and ions can pass through. Large quantities of water and ions (108 ions per second) are transported in large through the channels. The channel pores do not remain open all the time and many of them have proteins which act as gates which open and close in response to external signals. Such channels are called gated channels where a change in the three-dimensional conformation of the protein results in opening or closure of the pores. The “gates” may open in response to either: i) membrane potentials of magnitude. Such channels are called electrically gated channels; or ii) modulating signals like hormones, light or even phosphorylation. Many channel proteins are believed to be inducible as they are synthesized only in the presence of sufficient solute. The presence of K+, Cl-, and Ca2+ channels has been firmly established whereas there are indications of the presence of many more for other inorganic and organic ions. K+ channels have been established in the guard cells (see Fig. 4.7). You have already read about the mechanism of stomatal opening and closure in Unit 2. It is now believed that a variety of different channels may exist for a common ion, but operative under different voltage ranges, or induced by different signals. This means that the permeability of a membrane will be variable from time to time depending upon the activity of these ion channels. + Fig. 4.7: a) A K -channel showing a polypeptide chain of subunits; b) The pore- forming region is in the form of a loop between helices 5 and 6 (From Taiz et al). 107 Block 1 Water and Mineral Nutrition Selective accumulation of ions by roots Roots are capable of accumulating selective nutrients/ions. This is on account of the presence of specific transport proteins which are selective, and, in many cases, there is active transport. As a result, many a times the concentration of certain selected ions becomes more than that in the surrounding medium. The difference in the ion concentration within and outside the cell is called accumulation ratio. For example, K+ concentration within the maize root cells may be 1000 times more than the concentration outside the cell. On the other hand, Na+ is present in very low concentrations in plant cells (as against the animal cells). More so, Na+ gets actively expelled from most plants’ cells. As a result, the plant cells do not accumulate much Na+. Carriers (Transporters) Ionophores are organic molecules which act as carriers of specific ions. They increase the permeability of membranes to these specific ions, make them diffuse through the membrane and finally releasing them to the other side of the membrane. The free ionophore once again returns to its original site of the membrane to bind more such ions (Fig. 4.8). Such carriers are necessarily soluble in non-polar solvents. Fig. 4.8: Transportation of ions by carrier ionophores. Carrier proteins exhibit a high degree of specificity for a particular ion or organic metabolite (Fig. 4.9). Unlike the channels, their pores do not extend completely across the membrane. Their binding with a particular ion/molecule is reminiscent of an enzyme-substrate complex and the binding of the solute particles to the carriers induces a conformational change (a modification of the tertiary structure and shape) in the enzyme. They, however, do not induce any chemical modification of the bound ions’ metabolites. Since the carrier- mediated transport involves conformational changes in the protein and its travel through to the other side the bilayer, the rate of transport is much slower than that through channels (as discussed above). To explain, whereas 100- 1000 molecules ions per second could be transported by carriers, the figure 108 could reach a few million at the same time by channels. Unit 4 Nutrient Transport Fig. 4.9: Three different possible modes of transport of ions and other molecules across the plasma membrane. Unlike the channel-mediated transport, movement of ions/metabolites through the carriers can either be passive (facilitated diffusion) when operative along/ down an electrochemical potential without the expenditure of ATP, or by secondary active transport. Studies on plants cell membrane suggest that inorganic ions can permeate through aqueous protein channels called permeases. The permeases are ion specific because the permeability of different ions varies. Most membranes are more permeable to K+ than to other ions. Ion uptake through specific carrier proteins can be fooled by similar ions. It seems probable that certain proteins transport more than one ion at the same site and so the ions compete with each other. For example, K+, Cs+, Rb+ are apparently transported at the same site, and Na+ and Li+ both are transported at the other site. But ions transported at different sites do not compete with each other; for instance, K+ does not compete with Na+. Ion transport in plants also takes place through ionophores (see Fig 4.8). As you know ionophores are small polypeptides and proteins that shield the charge of ions from hydrophobic environment of the membrane. Ionophores have been isolated from bacteria and fungi. When they are added to the artificial lipid bilayer, they increase the rate of diffusion of specific ions by as much as one million-fold. There is much indirect evidence for mobile carrier proteins in plant membranes. So far only sucrose carrier is identified. The sucrose carriers help in loading of sucrose into the phloem. Secondary active transport is an active transport which does not utilize ATP but instead uses energy stored in the proton motive force (pmf) or other ion gradients and operates by symport or antiport. Driving Force of Protein-mediated transport Let us now find out what is the driving force involved in protein mediated transport. Many membrane transport proteins allow specific solutes to move across the lipid bilayer. If the transported molecule is uncharged, then the difference in its concentration on the two sides of the membrane, that is its concentration gradient determines the direction of transport. However, if the solute to be transported carries a net charge (either negative or positive), then both its concentration gradient and the total electrical gradient across the membrane influence its transport. 109 Block 1 Water and Mineral Nutrition In fact, all plasma membranes have electric potentials (transmembrane potential) across them with inside of the cell more negative compared to the outside. This is due to active transport of ions particularly H+ ions out of the cell. This potential difference allows the entry of positively charged ions into the cell but opposes the entry of negatively charged ions. 4.3.3 Pumps and Active Transport Nernst equation Many ions/metabolites tend to accumulate inside the cell as their uptake is very rapid and is even against the predicted concentrations as calculated from For example, the Nernst equation. Nernst equation is an equation that relates the reduction Nernst equation for a potential of an electrochemical reaction. These transport processes continue univalent cation at solute intake even against the established concentration gradients. Such a 25°C can be transport process is called Active Transport. This is also a unidirectional expressed as : transport mediated by carrier proteins. Moreover, it does not occur C10 spontaneously. Active transport also occurs in the reserve direction, i.e., from ∆E1 = 59 mV log inside to the outside, to maintain the internal solute concentration. Active C12 transport can be visualized as a carrier protein mediated process occurring which suggests that against a concentration gradient and requiring an input of energy. The source Nernst potential of 59 of metabolic energy source may not always be ATP although it is usually so. mV (C0/C1 = 10/1; as log 10 = 1) When the protein carrier utilizes directly energy derived from ATP-hydrolysis, corresponds to a ten oxidation-reduction reactions or the absorption of light, it is called as primary fold difference in active transport and the membrane proteins carrying out this uphill transport concentration. In are called Pumps. These pumps transport ions like H+ or Ca2+ but some of other words, for them also transport large organic molecules. passive diffusion of Thus, the presence of many anions like NO3− , Cl , H2PO4- , and SO42- in higher an ion to occur, a − membrane potential of 59 mV would be concentrations indicates that they are taken up actively. On the other hand the + 2+ 2+ sufficient to maintain internal concentrations of cations like NO , Mg , and Ca is lower than the a ten fold predicted values based on Nernst equation suggesting that they can enter the concentration cell by diffusion but will have to be exported (transported out) by active means. gradient of that ion. Thus, the Nernst equation helps us to distinguish between active and passive Membrane potential is exhibited by all transport. You will find that the Fig. 4.10 summarizes the control of ion cells due to the concentrations by passive and active processes occurring in the plant cell asymmetry of cytosol and vacuole. distribution of ions at the two faces of a membrane. Fig. 4.10: Control of ionic concentrations in the cytosol and vacuole by active (solid arrows ) and passive (dashed arrows ) transport 110 processes (From Taiz et al). Unit 4 Nutrient Transport It is quite clear from the above that: Sodium ions are pumped actively out of the cytosol into the extracellular space and into the vacuole. Potassium ions are capable of active as well as passive transport under different conditions. Both the vacuole and the cytosol accumulate K+ passively. Active uptake of K+ takes place when the concentration of K+ outside the cell is low. To maintain the cytosolic pH near 7.0, the excess protons (H+) are pumped out of the cytosol actively. This is perhaps the reason that both the extracellular medium as well the vacuole are nearly 1-2 units more acidic than the cytosol. Ca2+ leaves both the vacuoles and cytosol by active transport. Anions enter the cell by active mechanism. Since K+ is present in highest concentrations in plant cells and there is a Goldman Equation simultaneous transport of many ions across a membrane, the diffusion potential can be more accurately calculated by the Goldman equation It is an equation that helps to predict the (modified from Nernst equation). diffusion potential The primary active transport involves the utilization of energy from ATP across a membrane. hydrolysis, or an oxidation reduction. Membrane proteins carrying out such a It is a function of the transport are called pumps. Most pumps transport H+ and Ca2+. Also, the concentrations and plasma membrane H+-ATPase generates the gradient of electrochemical permeabilities of all potential of H+ across the plasma membrane. those ions which pass by various Together, the proton gradient and the normal membrane potential (i.e., the transmembrane gradient of electrochemical potential for H+) are called as the proton motive mechanisms. force (PMF). Goldman equation is a modified version of The PMF tends to move protons back across the membrane and is the Nernst equation. primary source of energy for many plant activities including ATP synthesis in the mitochondria and chloroplasts. This electrogenic H+ transport-generated proton motive force acts as a driving force for transporting various solutes actively (e.g., anions, cations, amino acids, and sugars) against their gradient of electrochemical potential. This is achieved by coupling the uphill transport of one solute to the downhill transport of another (Fig. 4.11). Fig. 4.11: Secondary active transport Symport and Antiport) coupled to the force created by the proton gradient (From Taiz et al