Baixe Mineral nutrition of higher plants e outras Manuais, Projetos, Pesquisas em PDF para Agronomia, somente na Docsity! Preface to First Edition Mineral nutrients are essential for plant growth and development. Mineral nutrition of plants is thus an area of fundamental importance for both basic and appUed science. Impressive progress has been made during the last decades in our understanding of the mechanisms of nutrient uptake and their functions in plant metabolism; at the same time, there have also been advances in increasing crop yields by the supply of mineral nutrients through fertilizer application. It is the main aim of this textbook to present the principles of the mineral nutrition of plants, based on our current knowledge. Although emphasis is placed on crop plants, examples are also presented from noncultivated plants including lower plants in cases where these examples are considered more suitable for demonstrating certain principles of mineral nutrition, either at a cellular level or as particular mechanisms of adaptation to adverse chemical soil conditions. Plant nutrition as a subject is closely related to other disciplines such as soil science, plant physiology and biochemistry. In this book, mineral nutrients in soils are treated only to the extent considered necessary for an understanding of how plant roots acquire mineral nutrients from soils, or how roots modify the chemical soil properties at the soil-root interface. Fundamental processes of plant physiology and biochemistry, such as photosynthesis and respiration, are treated mainly from the viewpoint of how, and to what extent, they are affected or regulated by mineral nutrients. Crop physiology is included as an area of fundamental practical importance for agriculture and horticul- ture, with particular reference to source-sink relationships as affected by mineral nutrients and phytohormones. Mineral nutrition of plants covers a wide field. It is therefore not possible to treat all aspects with the detail they deserve. In this book, certain aspects are covered in more detail, either because they have recently become particularly important to our under- standing of mineral nutrition, or because many advances have been made in a particular area in the last decade. Naturally, personal research interests and evaluation are also factors which have influenced selection. Particular emphasis is placed on short- and long-distance transport of mineral elements, on source-sink relationships, and on plant-soil relationships. It is also the intention of this book to enable the reader to become better acquainted with the mechanisms of adaptation of plants to adverse chemical soil conditions. The genetical basis of mineral nutrition is therefore stressed, as well as the possibilities and limitations of "fitting crop plant to soils", especially in the tropics and subtropics. One year after the second edition of his Mineral Nutrition of Higher Plants was pub- Ushed, Horst Marschner died in September 1996. He had contracted malaria during a visit to agricultural research projects in West Africa. Horst Marschner was bom in 1929. After an apprenticeship on a farm, he studied Agriculture and Chemistry at Jena University, and worked at Hohenheim University and the Technical University in Berlin. From 1977, he was a Professor at the Institute of Plant Nutrition, Stuttgart-Hohenheim. In addition, Horst Marschner went to California and to Australia for sabbatical terms, was responsible for field projects in many developing coimtries, and was visited in Hohenheim by guest scientists from all over the world who enjoyed his enthusiasm. In his research, he was interested in many subjects from soil science to plant physiology. He contributed specifically to the understanding of uptake and utilization of mineral nutrients by plants, rhizo- sphere effects, environmental aspects of plant nutrition, and plant adaptation to low nutrient supply and adverse soil conditions. He became a leading figure in plant mineral nutrition, and believed that science and rational thinking should be used to improve hxmian living conditions. In addition to writing several himdred scientific publications, Horst Marschner was a dedicated teacher and mentor of young scientists. We know of many people who enjoyed his contributions at meetings and conferences. Research in plant nutrition was fascinating to him, and he transmitted this fascination to those around him. His close involvement with practical experimentation was the basis for the clarity in his conmiunications. When asked to pubUsh a book on mineral nutrition of plants, Horst Marschner knew the risks and challenges of writing as the smgle author of a general textbook. He was gratified by the success of Mineral Nutrition of Higher Plants, the very positive conunents of his colleagues, and the world-wide appreciation by scientists and students. Close to retirement from his official duties as Head of the Institute, he was eagerly looking forward to spending more time discussing and presenting progress in his field, the science of plant nutrition. Now, after his unexpected death, this textbook has to serve as a legacy to an energetic, kind, constructive, and stimu- lating man. Stuttgart-Hohenheim Eckhard George March 1997 Volker R5mheld Glossary of Plant Species Botanical names of plant species frequently cited in this book. Alder Alfalfa Almond Apple Apricot Artichoke Aubergine Barley Bean (Common bean, snap bean) Beech Black gram Black walnut Blue lupin Broccoli Brussels sprout Cabbage Carrot Cassava Castor bean Cauliflower Chickpea Chinese cabbage Celery Coffee Common bean Cotton Couchgrass Cowpea Cucumber Alnus glutinosa (L.) Gaertn. Medicago sativa L. Amygdalus communis L. Malus sylvestris Mill. Prunus armeniaca L. Cynara scolymus L. Solanum melongena L. Hordeum vulgare L. Phaseolus vulgaris L. Fagus sylvatica L. Vigna mungo (L.) Hepper Juglans nigra L. Lupinus angustifolius L. Brassica oleracea L. convar. botrytis var. italica Plenck Brassica oleracea var. gemmifera L. Brassica oleracea L. var. capitata Daucus carota L. ssp. sativus (Hoffm.) Arcang. Manihot esculenta Crantz Ricinus communis L. Brassica oleracea L. convar. botrytis var. botrytis Cicer arietinum L. Brassica chinensis L. Apium graveolens L. var. rapaceum (Mill.) Coffea ssp. Phaseolus vulgaris L. Gossypium hirsutum L. Agropyron repens L. Vigna unguiculata (L.) Walp. Cucumis sativus L. XIV Glossary Douglas fir Faba bean Grapevine Groundnut Italian ryegrass Kallar grass Kentucky bluegrass Leek Lentil Lettuce Leucaena Loblolly pine Maize Mango Maritime pine Melon Mung bean Norway spruce Oat Oil palm Onion Pea Peach Peanut Pearl millet Pigeon pea Poppy Potato Pumpkin Rape Red beet Red clover Red pepper Reed Rice Rhodes grass Rye Ryegrass Scots pine Sesbania Shortleaf pine Sitka spruce Soybean Stinging nettle Strawberry Sugar beet Pseudotsuga menziesii (Mirb.) Franco ViciafabaL. Vitis vinifera L. ssp. vinifera Arachis hypogaea L. Lolium multiflorum Lam. Leptochloa fusca L. Kunth. Poa pratensis L. Allium porrum L. Lens culinaris L. Lactuca sativa L. Leucaena leucocephala Pinus taeda L. Zea mays L. Mangifera indica L. Pinus pinaster Soland in Ait. Cucumis melo L. Phaseolus mungo L. Picea abies (L.) Karst. Avena sativa 1.. Elaeis guineensis Jacq. Allium cepa L. Pisum sativum L. Prunus persica L. Batsch. Arachis hypogaea L. Pennisetum glaucum (L.) R.Br.s.l. Cajanus cajan L. Huth. Papaver somniferum L. Solanum tuberosum L. Cucurbita pepo L. Brassica napus L. var. napus Beta vulgaris L. ssp. vulgaris var. conditiva Alef. Trifolium pratense L. Capsicum annuum L. Phragmites communis Trinius Oryza sativa L. Chloris gay ana Kunth. Secale cereale L. Lolium perenne L. Pinus sylvestris L. Sesbania sesban Pinus echinata Mill. Picea stichensis Bong. Carr. Glycine max (L.) Merr. Urtica dioica L. Fragaria vesca Beta vulgaris L. ssp. vulgaris Mineral Nutrition of Higher Plants Table 1.1 Discovery of the Essentiality of Micronutrients for Higher Plants Element Iron Manganese Boron Zinc Copper Molybdenum Chlorine Nickel Year 1860 1922 1923 1926 1931 1938 1954 1987 Discovered by J. Sachs J. S. McHargue K. Warington A. L. Sommer and C. B. Lipman C. B. Lipman and G. MacKinney D. I. Amon and P. R. Stout T. C. Broyer et al. P. H. Brown et al. The term essential mineral element (or mineral nutrient) was proposed by Arnon and Stout (1939). These authors concluded that, for an element to be considered essential, three criteria must be met: 1. A given plant must be unable to complete its Hfe cycle in the absence of the mineral element. 2. The function of the element must not be replaceable by another mineral element. 3. The element must be directly involved in plant metabolism - for example, as a component of an essential plant constituent such as an enzyme - or it must be required for a distinct metaboUc step such as an enzyme reaction. According to this strict definition those mineral elements which compensate for the toxic effects of other elements or which simply replace mineral nutrients in some of their less specific functions, such as maintenance of osmotic pressure, are not essential, but can be described as 'beneficial' elements (Chapter 10). It is still difficult to generalize when discussing which mineral elements are essential for plant growth. This is particularly obvious when higher and lower plants are compared (Table 1.2). For higher plants the essentiaUty of 14 mineral elements is well estabhshed, although the known requirement for chlorine and nickel is as yet restricted to a limited number of plant species. Table 1.2 Essentiality of Mineral Elements for Higher and Lower Plants Classification Element Higher plants Lower plants Macronutrient Micronutrient Micronutrient and 'beneficial' element N, P, S, K, Mg, Ca Fe, Mn, Zn, Cu, B, Mo, CI, Ni Na, Si, Co i ,v + + ± — 4- (Exception: Ca for fungi) + (Exception: B for fungi) ± ± Because of continuous improvements in analytical techniques, especially in the purification of chemicals, this list might well be extended to include mineral elements that are essential only in very low concentrations in plants (i.e., that act as micro- nutrients). This holds true in particular for sodium and silicon, which are abundant in Introduction, Definition, and Classification of Mineral Nutrients 5 the biosphere. The essentiality of these two mineral elements has been established for some higher plant species (Chapter 10). Most micronutrients are predominantly constituents of enzyme molecules and are thus essential only in small amounts. In contrast, the macronutrients either are constituents of organic compounds, such as proteins and nucleic acids, or act as osmotica. These differences in function are reflected in the average concentrations of mineral nutrients in plant shoots that are sufficient for adequate growth (Table 1.3). The values can vary considerably depending on plant species, plant age, and concentration of other mineral elements. This aspect is discussed in Chapters 8 to 10, Table 1.3 Average Concentrations of Mineral Nutrients in Plant Shoot Dry Matter that are Sufficient for Adequate Growth"* Element Molybdenum Nickel^ Copper Zinc Manganese Iron Boron Chlorine Sulfur Phosphorus Magnesium Calcium Potassium Nitrogen ^From Epstein (1965). ^Based on Brown et al Abbreviation Mo Ni Cu Zn Mn Fe B CI S P Mg Ca K N . (1987b). //molg ^ dry wt 0.001 -0.001 0.10 0.30 1.0 2.0 2.0 3.0 30 60 80 125 250 1000 mgkg ^ (ppm) 0.1 - 0 . 1 6 20 50 100 20 100 — — — — — — % — — — — — — — 0.1 0.2 0.2 0.5 1.0 1.5 Relative number of atoms 1 1 100 300 1000 2000 2000 3000 30000 60000 80000 125000 250000 1000000 Ion Uptake Mechanisms of Individual Cells and Roots: Short-Distance Transport 2.1 General As a rule there is a great discrepancy between the mineral nutrient concentration in the soil or nutrient solution, on the one hand, and the mineral nutrient requirement of plants, on the other. Furthermore, soil and also in some cases nutrient solutions may contain high concentrations of mineral elements not needed for plant growth. The mechanisms by which plants take up nutrients must therefore be selective. This selectivity can be demonstrated particularly well in algal cells (Table 2.1), where the external and internal (cell sap) solutions are separated by only two membranes: the plasma membrane and the tonoplast. In Nitella the concentration of potassium,^ sodium, calcium, and chloride ions is higher in the cell sap than in the pond water, but the concentration ratio differs considerably between the ions. In Valonia growing in highly saHne seawater, on the other hand, only potassium is much more concentrated in the cell sap, whereas the sodium and calcium concentrations remain at a lower level in the cell sap than in the seawater. Although usually less dramatic, selectivity of ion uptake is also a typical feature of Table 2.1 Relationship between Ion Concentration in the Substrate and in the Cell Sap of Nitella and Valonia^ Nitella concentration (mM) Valonia concentration (mM) Ion Potassium Sodium Calcium Chloride A, Pond water 0.05 0.22 0.78 0.93 B, Cell sap 54 10 10 91 Ratio B/A 1080 45 13 98 A, Seawater 12 498 12 580 B, Cell sap 500 90 2 597 Ratio B/A 42 0.18 0.17 1 '̂ Modified from Hoagland (1948). Ion Uptake Mechanisms of Individual Cells and Roots Macropore Micropore Fig. 2.2 Schematic diagram of the pore system of the apparent free space. DFS, Donnan free space; WFS, water free space. In this network, a variable proportion of the pectins consist of polygalacturonic acid, originating mainly from the middle lamella. Accordingly both in roots and in the cell wall continuum of other plant tissue, the so-called apoplasm, the carboxyUc groups (R-COO~) act as cation exchangers. In roots therefore cations from the external solution can accumulate in a nonmetabolic step in thQ free space, whereas anions are 'repelled' (Fig. 2.2). Because of these negative charges the apoplasm does not provide a free space for the movement of charged solutes, and Hope and Stevens (1952) introduced the terms apparent free space (AFS). This comprises the water free space (WFS), which is freely accessible to ions and charged and uncharged molecules, and the Donnan free space (DFS), where cation exchange and anion repulsion take place (Fig. 2.2). Ion distri- bution within the DFS is characterized by the typical Donnan distribution which occurs in soils at the surfaces of negatively charged clay particles. Divalent cations such as Câ "̂ are therefore preferentially bound to these cation-exchange sites. Plant species differ considerably in cation-exchange capacity (CEC), that is, in the number of cation- exchange sites (fixed anions; R • COO"), located in cell walls, as shown in Table 2.3. As a rule, the CEC of dicotyledonous species is much higher than that of monocotyle- donous species. The effective CEC decreases as the external pH falls (Allan and Jarrell, 1989), and is usually much lower in intact roots than the values shown in Table 2.3. Table 2.3 Cation Exchange Capacity of Root Dry Matter of Different Plant Species^ Cation exchange capacity Plant species meq (100 g)~^ dry wt Wheat 23 Maize 29 Bean 54 Tomato 62 "Based on Keller and Deuel (1957). 10 Mineral Nutrition of Higher Plants Table 2.4 Uptake and Translocation of Zinc by Barley Plants'" Zinc supplied as^ ZnS04 ZnEDTA Rate of uptake and translocation (jug Zn g~^ dry wt per 24 h) Roots Shoots 4598 305 45 35 "Based on Barber and Lee (1974). ^Concentration of zinc in nutrient solution: 1 mg 1"^ Because of spatial limitations (Casparian band and exodermis; Section 2.5,1) only part of the exchange sites of the AFS are directly accessible to cations from the external solution. Nevertheless, the differences shown are typical of those that exist between plant species. Exchange adsorption in the AFS of the apoplasm is not an essential step for ion uptake or transport through the plasma membrane into the cytoplasm. Nevertheless, the preferential binding of di- and polyvalent cations increases the concentration of these cations in the apoplasm of the roots and thus in the vicinty of the active uptake sites at the plasma membrane. As a result of this indirect effect, a positive correlation can be observed between the CEC and the ratio of Câ "" to K"" contents in different plant species (Crooke and Knight, 1962; Haynes, 1980). Effective competition between H"̂ or mono- and polyvalent aluminium species or both with magnesium for binding sites in the apoplasm of roots is obviously a main factor responsible for the depression in magnesium uptake and appearance of magnesium deficiency in annual (Rengel, 1990; Tan et al., 1991) and forest tree species (Marschner, 1991b) grown on acid mineral soils (Section 16.3). The importance of cation binding in the AFS for uptake and subsequent shoot transport is also indicated by experiments with the same plant species but with different binding forms of a divalent cation such as zinc (Table 2.4). When zinc is supplied in the form of an inorganic salt (i.e., as free Zn^^), the zinc content not only of the roots but also of the shoots is several times higher than when zinc is supplied as a chelate (ZnEDTA), that is, without substantial binding of the solute in the AFS. In addition, restricted permeation of the chelated zinc within the pores of the AFS may be a contributing factor. Using these differences in uptake rate between metal cations like Zn̂ "̂ (and also Cu^+ and Mn̂ "̂ ) and their complexes with synthetic chelators in so- called chelator-buffered solutions calculations can be made of the concentrations of free metal cations in the external solution required for optimal plant growth (Bell et al, 1991; Laurie et al, 1991). According to these calculations, extremely low external concentrations at the plasma membrane of root cortical cells appear to be adequate to meet plant demand for these micronutrient cations (Section 2.5.4). With heavy-metal cations in particular, binding in the apoplasm can be quite specific. Copper, for example, may be bound in a nonionic form (coordinative binding) to nitrogen-containing groups of either glycoproteins or proteins of ectoenzymes, such as phosphatases or peroxidases, in the cell wall (Harrison et al, 1979; Van Cutsem and Ion Uptake Mechanisms of Individual Cells and Roots 11 CO c\ 1 2 D" 0 ^ 0. h^Ca x^ ̂-1 - E ^'K \ / A E 0 10 30 50 Time (min) 10 30 50 Time (min) Fig. 2.3 Time course of influx (I) and efflux (E) of "̂ Ĉa and "̂ K̂ in isolated barley roots. After 30 min (arrow) some of the roots were transferred to solutions with nonlabelled Ca^^ and K^. The proportion of the exchangeable fraction in the apparent free space is calculated by extrapolation to zero time (x). Gillet, 1982). This cation binding in the apoplasm can contribute significantly to the total cation content of roots, as shown by studies of the uptake of polyvalent cations such as copper, zinc, and iron. This is also demonstrated by the data in Table 2.4. When supplied in non-chelated form high contents of polyvalent cations in the roots compared to the shoots therefore not necessarily reflect immobilization in the cytoplasm or vacuoles but may result from preferential binding in the apoplasm of the root cortex. The root apoplasm may also serve as transient storage pool for heavy metals such as iron and zinc which can be mobilized, for example by specific root exudates such as phytosiderophores, and translocated subsequently into the shoots (Zhang etal,, 1991b, c). The size of this storage pool for iron probably plays a role for genotypical differences in sensitivity to iron deficiency in soybean (Longnecker and Welch, 1990). On the other hand, excessive uptake of calcium may be restricted by precipitation as calcium oxalate in the cell walls of the cortex (Fink, 1992b). 2.2.2 Passage into the Cytoplasm and the Vacuole Despite some selectivity for cation binding in the cell wall (Section 2.2.1), the main sites of selectivity in the uptake of cations and anions as well as solutes in general are located in the plasma membrane of individual cells. The plasma membrane is an effective barrier against the diffusion of solutes either from the apoplasm into the cytoplasm (influx) or from the cytoplasm into the apoplasm and the external solution (efflux). The plasma membrane is also the principal site of active transport in either direction. The other main barrier to diffusion is the tonoplast (vacuolar membrane). In most mature plant cells the vacuole comprises more than 80-90% of the cell volume (Leigh and Wyn Jones, 1986; Wink, 1993; also see Fig. 2.1) acting as central storage compartment for ions, but also for other solutes (e.g. sugars, secondary metabolites). It can be readily demonstrated that the plasma membrane and the tonoplast function as effective barriers to diffusion and exchange of ions, as shown for example, for K"̂ and Ca "̂̂ (Fig. 2.3). Most of the Ca^"" (^^Ca) taken up within 30 min (influx) is still readily exchangeable (efflux) and is almost certainly located in the AFS. In contrast only a 14 Mineral Nutrition of Higher Plants CH^O-P-0-CH2-CH2-N^(CH3) R ̂ / N / V ' ^ . / ^ A . / V ^ 0-CH O" Phosphatidylcholine (lecithin) CH2OH • o J \ H 01^0 OH OH H Monogalactosyl diglyceride (Long chain polyunsaturated fatty acids) 9H2- R̂ / \ / V ^ V \ y v ^ v / ^ o - C H D 2 • o J \ H Oh^c o C H 2 - § - 0 ' "" .H O OH OH H Sulfoquinovosyl diglyceride Another important group of membrane lipids consists of sterols, for example p- sistosterol: B-Sistosterol Through their structural role in membranes sterols may indirectly affect transport processes such as the activity of the proton pumping ATPase in the plasma membrane (Sandstrom and Cleland, 1989). In agreement with this assumption the sterol content is very low in endomembranes (e.g. endoplasmic reticulum) but may make up more than 30% of the total lipids in the plasma membrane (Brown and DuPont, 1989) and also in the tonoplast (Table 2.6). Despite these differences in lipids, the fatty acid composition of the phospholipids is similar in both membranes. The long-chain fatty acids in polar membrane lipids vary in both the length and degree of unsaturation (i.e. number of double bounds) which influence the melting point (Table 2.6). Lipid composition not only differs characteristically between membranes of indi- vidual cells but also between cells of different plant species (Stuiver et al., 1978), it is also strongly affected by environmental factors. In leaves, for example, distinct annual variations in the levels of sterols occur (Westerman and Roddick, 1981) and in roots both phospholipid content and the proportion of highly unsaturated fatty acids decrease Ion Uptake Mechanisms of Individual Cells and Roots 15 Table 2.6 Lipid and Fatty Acid Composition of Plasma Membranes and Tonoplasts from Mung Bean"" Lipids Phospholipids Sterols Glycolipids Fatty acid Palmitic acid Stearic acid Oleic acid Linoleic acid Linolenic acid Others Plasma membrane jumol mg~^ protein L29 L15 0.20 Fatty acid composition of the phospholipids Chain length Ci6 ^ 1 8 ^18:1 M8:2 M8:3 Melting point (°C) +62.8 +70.1 + 13.0 -5 .5 -11.1 — Plasma membrane (% of total) 35 6 9 21 19 10 Tonoplast jumol mg"^ protein 1.93 1.05 0.80 Tonoplast (% of total) 39 6 9 22 20 4 "Based on Yoshida and Uemura (1986). Reprinted by permission of the American Society of Plant Physiologists. ^Numeral to the right of the colon indicates the number of double bounds. under zinc deficiency (Cakmak and Marschner, 1988c). In many instances changes in lipid composition reflect adaptation of a plant to its environment through adjustment of membrane properties. Generally, highly unsaturated fatty acids predominate in plants that grow in cold climates. During acclimatization of plants to low temperatures an increase in highly unsaturated fatty acids is also often observed (Bulder et aL, 1991). Such a change shifts the freezing point (i.e. the transition temperature) of membranes to a lower temperature and may thus be of importance for maintenance of membrane functions at low temperatures. It is questionable, however, to generalize about the effect of temperature on lipid composition of membranes. In rye, for example, which is a cold-tolerant plant species, the proportion of polyunsaturated fatty acids in the roots decreased rather than increased as the roots were cooled (White et aL, 1990b). During acclimatization of roots to low temperatures synthesis of new membrane proteins is also enhanced (Mohapatra etal., 1988) and phospholipids increase consider- ably (Kinney et al., 1987). Since phospholipids probably act as receptors for phytohor- mones such as gibberellic acid, increasing responsiveness of membranes to gibberellic acid at low temperatures may be related to these changes (Singh and Paleg, 1984). The property of membranes in ion selectivity and lipid composition are often highly correlated as for example between chloride uptake and sterols (Douglas and Walker, i983) and galactolipids (Section 16.6). Also the crop plant species bean, sugar beet and barley differ not only in the fatty acid composition of root membranes (Stuiver et al., 1978) but also considerably in the uptake of sodium (Section 10.2). Alterations in the lipid composition of root membranes are also typical responses to changes in the mineral nutrient supply or exposure to salinity (Kuiper, 1980). Of the 16 Mineral Nutrition of Higher Plants mineral nutrients, calcium plays the most direct role in the maintenance of membrane integrity, a function which is discussed in Section 2.5.2. In soybean roots, changes in calcium and nitrogen supply affect the ratio of saturated to unsaturated fatty acids as well as the uptake rate of certain herbicides (Rivera and Penner, 1978). An increase in membrane permeability can be observed in roots suffering from phosphorus deficiency (Ratnayake et aL, 1978) and zinc deficiency (Welch et al., 1982; Cakmak and Marschner, 1988b, 1990). In the case of phosphorus deficiency, a shortage of phospho- hpids in the membranes has been assumed to be the responsible factor. In the case of zinc deficiency, autoxidation in the membranes of highly unsaturated fatty acids is presumably involved in membrane leakiness (Section 9.4). The dynamic nature of membranes is clearly demonstrated, for example, by the rapid decrease in efflux of low-molecular-weight solutes (potassium, sugars, amino acids) after resupplying of zinc to zinc-deficient roots (Cakmak and Marschner, 1988b). Another example is the rapid incorporation of externally supplied membrane constitu- ents such as phospholipids into the membrane structure. For the plasma membrane turnover rates seem to be in the order of only a few hours (Steer, 1988a). Such high turnover rates indicate that certain subunits (e.g. with intrinsic proteins; Fig. 2.4) are already synthesized and transported to the plasma membrane via secretory vesicles as for example of the Golgi apparatus (Coleman et al., 1988). The incorporation also of externally supplied compounds, however, renders mem- branes more sensitive to injury. The incorporation of antibiotics such as nystatin induces the formation of pores ('holes') in the membranes and a corresponding rapid leakage of low-molecular-weight solutes such as potassium. Monocarboxylic acids such as acetic acid and butyric acid, also induce membrane injury. The undissociated species of these acids are readily taken up and lead to a sharp rise in membrane leakiness, as indicated by the leakage of potassium and nitrate from the root tissue (Lee, 1977). The capacity of monocarboxylic acids to induce membrane leakiness increases with the chain length of the acids [C2 (acetic acid) -^ Cg (caprylic acid)] and hence with increased lipophilic behaviour, as well as with a lowering of the external pH (R • COO~ + H^ ^ R • COOH). Undissociated monocarboxylic acids may increase membrane leakiness by changing the fatty acid composition of membranes, particularly by decreasing the proportion of polyunsaturated fatty acids such as linolenic acid (Jackson and St. John, 1980). The effect of monocarboxylic acids on the membrane permeability of roots is of considerable ecological importance, since these acids accumulate in waterlogged soils (Section 16.4). These examples demonstrate that composition, structure and integrity of membranes are affected by a range of environmental factors. In the last decade in particular increasing evidence has accumulated that a range of environmental stress factors such as high fight intensity, drought, chilling, air pollutants, and also mineral nutrient deficiencies are harmful to plants by impairment of membrane integrity, and that elevated levels of toxic oxygen species are causally involved in this impairment (Elstner, 1982; Hippeh and Elstner, 1991). As shown in a model in Fig. 2.5, these toxic oxygen species are either radicals such as superoxide (02*~) or hydroxyl (OH), or the molecule hydrogen peroxide (H2O2). All are formed in various reactions and metabolic processes where oxygen is involved, for example photosynthesis (Asada, 1992) and respiration, including oxidation of NADPH Ion Uptake Mechanisms of Individual Cells and Roots 19 Table 2.7 Respiratory Energy Costs for Ion Uptake in Roots of Carex diandra^ Proportion of total ATP demand required for Ion uptake Growth Maintenance of biomass 40 36 39 25 Plant age (days) 60 17 43 40 80 10 38 52 ^Based on Werf et al. (1988). proportion declines in favour of ATP demand for growth and maintenance of biomass. In principle, similar results have been found with maize (Werf et al., 1988). These calculations at a whole plant level on ATP demand for ion uptake by roots have to be interpreted with care with respect to energy demand for membrane transport of ions in root cells. Firstly, these calculations include energy demand for radial transport through the roots and secretion into the xylem (Sections 2.7 and 2.8). Secondly, a relatively large proportion of carbohydrates supplied from the shoot to the roots are oxidized via the nonphosphorylating mitochondrial electron transport chain ('alterna- tive pathway'; Section 5.3) yielding less ATP synthesized per molecule of carbohydrate oxidized. Taking this shift in respiratory pathway into account, a requirement of one molecule ATP per ion transported across the plasma membrane has been calculated (Lambers et al., 1981). Such calculations are based on net uptake and include energy requirement for re-uptake ('retrieval') of ions from the apoplasm of the root ('efflux costs') which are assumed to be in the range of 20% of the influx costs (Bouma and De Visser, 1993). Thirdly, a direct coupUng of ATP consumption and ion transport across membranes is the exception rather than the rule. As discussed below, ATP-driven pumps at the plasma membrane and the tonoplast also have functions other than transport of mineral nutrients and organic solutes across the membranes. 2.4.2 Active and Passive Transport: Electrogenic Pumps, Carriers, Ion Channels Solute transport across membranes is not necessarily an active process. Solutes may be more concentrated on one side of the membrane (i.e. they may possess more free energy) and thus diffuse from a higher to a lower concentration (or chemical potential). This 'downhill' transport across a membrane is, in thermodynamic terms, a passive transport with the aid of carriers, or across aqueous pores (Clarkson, 1977). In cells, such downhill transport of ions across the plasma membrane may be maintained by a lowering of the ion activity in the cytoplasm, for example, due to adsorption at charged groups (e.g., R • COO~ or R • NHs") or to incorporation into organic structures (e.g., phosphate into nucleic acids). This is particularly true in meristematic tissues (e.g., root tips). In contrast, membrane transport against the gradient of potential energy ('uphill') must be linked directly or indirectly to an energy-consuming mechanism, a 'pump' in 20 Mineral Nutrition of Higher Plants Microelectrode <2> V///////////7ZA External solution n Cytoplasm \//////////////A / Cell wall Vacuole V7///////////////////////ZZ77/A B Chemical equilibrium External solution 1 mMK* 1 mM c r Cell sap (vacuole) (-59 mV) — 1 mM K* — 1 mM c r Electrochemical 1 mM K* equilibrium i ^M CI" - lOmMK* 0.1 mM c r Fig. 2.7 A. Schematic presentation of the system for measuring electropotentials in plant cells. B. Example of the calculation of ion distribution at chemical and electrochemical equilibrium assuming an electropotential of -59 mV. the membrane. To determine whether an ion is actively transported across a mem- brane, however, both the activity or concentration of the ion on either side of the membrane (i.e., the chemical potential gradient) and the electrical potential gradient (i.e., differences in millivolts) across the membrane must be known. By means of microelectrodes inserted into the vacuoles, a strongly negative electrical potential can be measured between the cell sap and the external solution (Fig. 2.7). The first measurements of this kind were made in cells of giant algae such as Char a, where strongly negative electrical potentials of between -100 and -200 mV were found. The same method was used by Higinbotham et al. (1967) and Glass and Dunlop (1979) to demonstrate the existence of similar electrical potential gradients in cells of higher plants. The concentration at which cations and anions on either side of a membrane are in electrochemical equilibrium or which ions in the external solution are in equiHbrium with those in the vacuole can be calculated according to the Nernst equation: E (mV) = - 5 9 log concentration inside (vacuole) concentration outside (external solution) According to this equation, at a negative electropotential of - 5 9 mV, monovalent cations such as K^ or anions such as CI" would be in electrochemical equilibrium if their concentration in the vacuole were 10 times higher (K"^) or 10 times lower (CP) than in the external solution (Fig. 2.7). For divalent cations or anions the difference between chemical and electrochemical equilibrium differs by even more than the factor of 100. In cells of higher plants the electrical potential differences between vacuoles and the external solution are generally higher than - 5 9 mV (Table 2.8). Thus, as a rule, in terms of electrophysiology, only anion uptake into the vacuoles would always require an active transport process. This is indicated in Table 2.8 in the differences between the Ion Uptake Mechanisms of Individual Cells and Roots 21 Table 2.8 Experimentally Determined and Calculated Ion Concentration (mM) According to the Electrical Potential Differences in Roots of Pea and Oat" Ion Potassium Sodium Calcium Chloride Nitrate Pea roots (- Experimental 75 8 2 7 28 -110 mV) Calculated 74 74 10800 0.014 0.027 Oat roots (- Experimental 66 3 3 3 56 -84 mV) Calculated 27 27 1400 0.038 0.076 "Composition of the external solution: 1 mM KCl, 1 mM Ca(N03)2, and 1 mM NaH2P04. Based on Higinbotham er fl/. (1967). anion concentrations in the vacuoles based on calculations according to the electro- chemical equilibrium and the anion concentrations in the vacuoles actually found in the experiment. In this example, the only cation that would require active transport for its uptake is K"̂ in oat roots. At low external K^ concentration, active transport is usually required (Cheeseman and Hanson, 1979). For Na"^ and Câ "̂ in particular, the equilibrium concentration in the cell sap (i.e., the calculated ones) would be much higher than that found experimentally in the steady state (Table 2.8). A possible explanation for this discrepancy is that the plasma membrane strongly restricts permeation by these ions or that the ions are pumped (transported) back into the external solution. For Na^ such an efflux pump at the plasma membrane of root cells has been estabhshed for various plant species (Jacoby and Rudich, 1985; Schubert and Lauchh, 1988). For Câ "̂ active extrusion (Ca^"^ efflux pump) at the plasma membrane also exists in root cells (Gibe and Sommarin, 1991). The general importance of this Câ "̂ efflux pump at both plasma membrane and tonoplast for the functioning of cells is discussed in Section 8.6. Since the Ca^^ concentrations in soil solutions are usually higher than 1 mM, a Câ "̂ efflux pump to prevent Câ "̂ transport along the electrochemical gradient (e.g., Table 2.8) would have a considerable energy requirement. It is likely, therefore, that additional physicochemical factors such as the size and charge of Ca^^ strongly restrict permeation along the electrochemical potential gradient across the plasma membrane. In recent years impressive progress has been made in understanding both the mechanisms leading to the formation of electropotentials across membranes and the importance of these potentials for cell growth and functioning. Progress was possible by new techniques allowing measurements of membrane potentials and ion fluxes in isolated membrane vesicles or in sections of membranes (patch clamp technique, Hedrich and Schroeder, 1989). Soipe of the principles of ion transport in membranes are shown in Fig. 2.8. An ATP-driven H"̂ pump ('proton motive force'; Poole, 1978) transports H"̂ through the membrane from the internal to the external surface, for example in the plasma membrane from the cytoplasm to the apoplasm, thereby creating a gradient in pH and electropotential. Transport of cations and anions along the gradient is mediated either by ion selective carriers or channels. This model also takes 24 Mineral Nutrition of Higher Plants Table 2.9 Some Characteristics of the Proton Pumps in the Tonoplast ATPase PPjase H"^pumping activity'' (mol H"̂ m"^ s~ )̂ 214 95 Activity affected^ Stimulated by Mg^^, CI" Mg^^, K^̂ , NOi" Not affected or inhibited by K"*̂ , NO3- CP "From Hoffmann and Bentrup (1989). ^Compiled data from Bennet et al. (1984); Marquardt and Luttge (1987) and Pugliarello et al. (1991). cotransport at the plasma membrane of root cells has been presented for chloride (Ullrich and Novacky, 1990), phosphate (Dunlop, 1989) and nitrate (McClure et al, 1990a), as well as for amino acids (Petzold et al, 1989). The stoichiometry of this cotransport is not yet clear; more than one proton may be transported per negative charge of the anion (Lass and Ullrich-Eberius, 1984; McClure et al, 1990b). There are also other views on the mechanisms of anion transport across the plasma membrane. According to Liu (1979), phosphate uptake by corn roots is mediated by OH~/phosphate countertransport in which the downhill transport of OH~ from the high electrochemical potential in the cytoplasm (high pH and strong negative charge) into the apoplasm is coupled with a countertransport of phosphate anions into the cyto- plasm. However, experimental evidence for exchange or transport of OH~ or HCO3, across membranes as driving force for anion transport is weak, as specific inhibitors of OH~ and HCO^ transport are not available (Kurdjian and Guern, 1989). At the tonoplast the existence of two functionally and physically distinct proton pumps is now established, an H^-ATPase and an inorganic pyrophosphatase, PPjase (Fig. 2.9). Both proton pumps are phosphohydrolases using either ATP or inorganic pyrophosphate as energy source (Section 8.4). Magnesium is essential for both pumps, indicating that Mg • ATP and Mg • PPi are the substrates. Inorganic pyrophosphate is generated in several major biosynthetic pathways (Rea and Sanders, 1987) such as starch synthesis (Section 8.4) or activation of sulfate (Section 8.3.2). The concentration of PPi in the cytosol is assumed to be in the range of 50-390 pm which is adequate to drive this proton pump (Chanson et al., 1985). The relative contribution of the PPiase to the total proton pumping activity at the tonoplast seems to be lower than that of the ATPase, and both pumps are quite differently affected by inorganic cations and anions (Table 2.9). Except for the essentiality of Mg "̂̂ for both pumps, the Mg-ATPase is stimulated by chloride and inhibited (or not affected) by potassium and nitrate whereas the reverse is true for the Mg-PPjase. The H^ pumping Mg-PPjase exhibits an obligatory dependency on the presence of K^ and the K"̂ activation occurs at the cytoplasmic phase (Davies et al, 1991b). The implications of this difference in capacity and sensitivity of both pumps for ion accumulation in vacuoles ox radial transport of ions across the roots (Section 2.7) are not clear. Different proportions of activities of both pumps in the tonoplast.pf roots Ion Uptake Mechanisms of Individual Cells and Roots 25 may well be involved in the genotypical differences between plants in chloride translocation into the shoots (Chapter 16). The proton pumps at the tonoplast are required for maintenance of a high cytosoHc pH and simultaneously provide the driving force for cation transport into the vacuole as countertransport (or antiport, Fig. 2.9). This countertransport is not only important, for example, for turgor regulation (high vacuolar K"̂ concentrations, Section 8.7) but also for the maintenance of low cytosolic concentrations of sodium (Garbarino and DuPont, 1989; Section 16.6.5) and calcium (Chanson, 1991; Section 8.6). For anions the situation is less clear. Whereas in leaf cells of plants with crassulacean acid metabolism (CAM, Section 5.2.4) a stoichiometric proton-malate anion cotransport into the vacuoles has been demonstrated, for roots the evidence for coupling of such a proton- anion transport is weak. Transport of anions from the cytoplasm into the vacuole may follow the gradient in electropotential which is less negative in the vacuole as compared with the cytoplasm (Fig. 2.9). Whereas the existence of the two proton pumps at the tonoplast (ATPase; PPiase) and of the H^-ATPase at the plasma membrane is well established, it is still a matter of controversy whether a second system of proton translocation across the plasma membrane from the cytoplasm to the apoplasm is involved in the formation and maintenance of the transmembrane electropotential and pH (Fig. 2.9). This second system is linked to a redox chain with NAD(P)H as electron donor. There is good evidence for the involvement of this system in auxin-induced enhanced growth (Morre et al., 1988), in antimicrobial activity (Dahse et al., 1989), or in the reduction of Fe(III) at the surface of the root plasma membranes (Bienfait and Liittge, 1988). However, so far mainly ferricyanide or other artificial compounds have to be used as electron acceptors to achieve a substantial transmembrane proton transport capacity (Luster and Buckhout, 1988). The role of this redox system for transmembrane proton gradients and ion transport remains questionable until a physiological electron acceptor can be found (Serrano, 1989). In view of the effectivity of ascorbate (ascorbic free radical) as electron acceptor for the transmembrane redox pump (Gonzales-Reyes et al., 1992) and the relatively high ascorbate concentrations in the apoplasm of leaves (PoUe et al., 1990) a role of this system in leaves in plasma membrane transport of ions and other solutes cannot be excluded (Chapter 3). More recently, the existence of ion channels also in plant cell membranes has been established and, thus, channels are included in current models of membrane transport of ions (Figs. 2.8 and 2.9). Ion channels are unique among transport proteins in their ability to regulate or 'gate' ion flux subject to the physical-chemical environment of the channel protein (Blatt and Thiel, 1993). These channels permit rapid passive per- meation (uniport) of ions through membranes. Open channels catalyze the permeation of 10̂ to 10̂ ions per second which is at least three (Tester, 1990) or even five (Bentrup, 1989) orders of magnitude faster than carrier-mediated transport of ions. However, ion channels are closed most of the time, and their number per cell seems to be rather small. For example, in the plasma membrane of leaf cells about 200 K^ channels per cell are assumed (Kourie and Goldsmith, 1992). So far, specific channels for K"̂ , Câ "̂ , H"̂ and Cl~ have been identified, and for NO^ a channel is postulated at the tonoplast (Tyerman, 1992). There are many assumptions about the function of these ion channels in plant cells. 26 Mineral Nutrition of Higher Plants They are important for osmoregulation, for example in guard cells of leaves (Schauf and Wilson, 1987) and in seismonastic and nyctinastic movements of leaves (Schroeder and Hedrich, 1989), i.e., in processes where rapid transport of low molecular solutes like K^ or Cl~ between cell compartments is required as a response to environmental signals (Section 8.7). Selective Câ "̂ channels in the plasma membrane and tonoplast leading to rapid increase in cytosolic free Câ "̂ concentration are considered of key importance for signal transduction and functioning of Câ "̂ as a secondary messenger in the cytoplasm by modulating enzyme activities (Briskin, 1990; Section 8.6.7). Besides these specific functions of ion channels their role in ion uptake, for example by roots, is not very clear. For the uptake of divalent cations in general and Câ "*" in particular, opening of the channels facilitates rapid influx into the cytoplasm of root cells. For K"̂ uptake an inward rectifying channel is proposed which opens upon hyperpolarization of the plasma membrane and faciUtates the K"̂ influx in presence of high external concentrations (>1 mM K^; Maathuis and Sanders, 1993; White, 1993). Another high conductance channel in the plasma membrane of root cells is permeable for both monovalent and divalent cations, opens upon depolarization (i.e. drop in membrane potential) and permits rapid influx of cations such as Câ "̂ but facilitates rapid efflux of K"̂ along the electrochemical potential gradient (outward rectifying K"̂ channel) at low (<1 mivf) external K"̂ concentrations (White, 1993). Thus, cation channels in the plasma membrane of root cells presumably play an important part in uptake of divalent cations even at low external concentrations but for monovalent cations and K"̂ in particular only at high external concentrations. Although channels in membranes allow rapid, passive fluxes of solutes, the dimen- sions of these channels are not suited for permeation of macromolecules. Nevertheless, macromolecules like proteins (e.g. insulin; Horn et al., 1990) or ferritin particles (Joachim and Robinson, 1984; Section 8.4.5) are also taken up by plant cells. Most probably, endocytosis (pinocytosis) is the responsible mechanism in which plasma membrane vesicles mediate the permeation. Interestingly, the uptake of insulin in plant cells via endocytosis requires a coupling of the protein with the vitamin biotin (Horn et al., 1990). This capacity for uptake of macromolecules reflects the dynamic properties of the membrane structures (Section 2.4.1). The importance of this uptake capacity in plants should not be overestimated, however, as the pores in the cell walls strongly restrict the permeation of macromolecules (Section 2.2.1). 2.4.3 The Kinetics of Transport As a rule ion uptake by plant cells and roots has features of a saturation kinetics. This is in accordance with the assumption of control, as for example by the number of binding sites of ions (carrier, permeases), or the capacity of the proton efflux pumps, in the plasma membrane and tonoplast (Section 2.4.2). The pioneering work of Emanuel Epstein and his group in the early 1950s contributed fundamentally to the better understanding of ion uptake and its regulation in plants by regarding the kinetics of ion transport through membranes of plant cells as formally equivalent to the relationship between an enzyme and its substrate, using terms of enzymology (Fig. 2.10). Com- paring a carrier to an enzyme molecule and the ion to the substrate for the enzyme, the transport rate of an ion is dependent on the following two factors: Ion Uptake Mechanisms of Individual Cells and Roots 29 Table 2.10 Effect of low Phosphorus Concentrations on Influx and Efflux of Phosphorus in Maize Roots'̂ P concentration supplied (JUM) 0.2 2.0 "Elliott etaL(19S4). (nmol P g Influx 0.21 4.40 Pflux ^ root fresh wt min~^) Efflux 0.15 0.32 Efflux (%) 71 7 Table 2.11 Influence of the Phosphorus Nutritional Status of Soybean Plants on Short- term Uptake Parameters of Phosphorus" Plants grown at P concentration (/^M) 0.03 0.3 3.0 30.0 P content Shoot 0.22 0.34 0.59 0.66 (% dry wt) Root 0.23 0.30 0.56 0.90 ^max ^ m (molcm-^s"^ X 10" i^) (juu) 17.6 1.6 16.9 1.7 6.5 1.2 3.7 1.0 ''Based on Jungk etal. (1990). for a high proportion - almost 40-50% of the influx - most probably relating to the high concentrations of nitrate and ammonium in the cytoplasm (Lee and Clarkson, 1986; Jackson et al., 1993). The rapid exchange between ions in the external solution and in the cytoplasm is reflected in the half-time for exchange (ti/2) which is for sulfate in the range of 10-20 min (Deane-Drummond, 1987) and for nitrate between 4 min (Lee and Clarkson, 1986) and 107 min (Macklon et al., 1990). These rates of exchange with the cytoplasmic pool are usually orders of magnitude higher than the rates of exchange with the ions in the vacuole (e.g. about 700 h for nitrate; Macklon et al., 1990). Because of both the high exchange rates and the small volume of the cytoplasmic compartment (--5% of the total cell volume in differentiated cells), the role of efflux for the net uptake can only be measured in short-term studies, usually with radioisotopes (e.g. ^^N; ^^P). In view of the current models on structure and functioning of the plasma membrane (Fig. 2.9), the relatively high efflux rates of ions may be related to either ion channels or proton-mediated transport from the cytoplasm into the apoplasm by cotransport (anions) or countertransport (cations). The parameters of ion-uptake kinetics are also strongly affected by the nutritional status of the plants. This holds true not only for Cmin but also for K^ and particularly ^max- An example for this is given in Table 2.11 for phosphorus. With increasing phosphorus content in the plants, K^^ slightly decreases and /max rapidly decreases. 30 Mineral Nutrition of Higher Plants indicating an effective feed-back regulation. As /max values were based on net uptake in this experiment, the contribution of increased efflux with higher internal phosphorus concentrations cannot be evaluated. At least for nitrate, however, measurements of influx clearly indicate that besides efflux other mechanisms contribute to the decline in net uptake when internal concentrations are high. In barley roots with increasing internal nitrate concentrations both /max ^^^ ^m decrease by a factor of 4-5, indicating an effective feedback regulation of the influx component (Siddiqi et al., 1990). Evaluations of the kinetic parameters of nitrate uptake are complicated by the fact that there are obviously two uptake and transport systems located in the plasma membrane.One is constitutive, with a low capacity system, and the other is a system that is inducible by nitrate and which has both, a higher affinity (lower K^) and higher transport capacity (higher /max) for nitrate (Behl et al, 1988; Siddiqi et al., 1990). Accordingly, both, Ĵ m and /max are quite different in noninduced as compared with induced plants (Wieneke, 1992). Inhibitors of protein synthesis strongly depress the formation of this inducible transport system (Wieneke, 1992) in which arginine groups seem to play an essential role in binding or transport of nitrate or both (Ni and Beevers, 1990). Some of the implications of the feedback regulation for mineral nutrient uptake are discussed in Section 2.5.6. In the high concentration range a linear relationship is often found between external concentrations (>lmM) and influx rate of ions, as for example rubidium (Kochian and Lucas, 1982) and nitrate (Siddiqi et al., 1990). It is quite likely that the linear relationships (formerly also defined as System II, Epstein, 1972) are reflections of passive ion fluxes in ion channels through the plasma membrane along the gradient in ion concentrations or activities. In view of the usually low ion concentrations in soil solutions, the ecological importance of these mechanisms operating in the high concentration range for the mineral nutrition of soil-grown plants may be questioned for potassium, but not for divalent cations (Section 2.4.2). Non-energized (passive) influx of ions through channels in the plasma membrane certainly plays an important role in saline soils (Section 16.6), in mineral element transport in plants, especially xylem loading in the roots (Section 2.8) and xylem unloading, i.e. uptake in ceUs along the pathway and in leaf cells (Section 3.2). 2.5 Characteristics of Ion Uptake by Roots 2.5.1 Influx into the Apoplasm Before reaching the plasma membrane of root cells ions have to pass through the cell walls. In general, movement of ions and other low-molecular-weight solutes by diffusion or mass flow is not restricted to the external surface of the roots, that is the rhizodermal cells (Fig. 2.1). The cell walls and water-filled intercellular spaces of the root cortex are also, at least to a certain extent, accessible to these solutes from the external solution. The main barrier to solute flux in the apoplasm of roots is the endodermis, the innermost layer of cells of the cortex (Fig. 2.12). In the radial and transverse walls of the endodermis, hydrophobic incrustations (suberin) - the Casparian band - constitute an Ion Uptake Mechanisms of individual Cells and Roots 31 CB = Casparian band ^ = Xylem B ::̂ :̂ Rhizodermis Root hairs Fig. 2.12 A. Cross section of a differentiated root zone of maize. B. Schematic presentation of cross section. effective barrier against passive solute movement into the stele. In most species of the angiosperms suberin lamellae are also found in the hypodermis, or exodermis (cell layer below rhizodermis; Peterson, 1988; Enstone and Peterson, 1992). The exodermis (Fig. 2.12) may also function as a barrier to protect the inner cortex from colonization by microorganisms, for example of sorghum roots by the endophyte Poly myxa sp. (Galamay^^fl/., 1992). Compared with the Casparian band the formation of this exodermis along the root axis is delayed, particularly in fast-growing roots (Peterson, 1988). There are different views on the effectiveness of the exodermis as a barrier against passive solute flux into the root apoplasm (Clarkson et al., 1987; Peterson, 1988; Section 2.7). In plants adapted to submerged conditions the exodermis serves another function, namely as an effective barrier against oxygen diffusion (leakage) from the root aerenchyma into the rhizosphere (Section 14.3). The volume of root tissue accessible for passive solute flux - the free space - represents only a small fraction - 5 % in maize (Shone and Flood, 1985) of the total root volume. The presence of this free space enables individual cortex cells to take up solutes directly from the external solution. For a given volume of the free space the extent of solute flux into the free space depends on various factors such as the rate of transpiration, solute concentration and root hair formation. There has been a tendency to overestimate the importance of the free space for ion uptake by roots. As shown more than 25 years ago by Vakhmistrov (1967), at low external concentration root hair formation is usually extensive and uptake, of potassium and phosphorus for example, is limited mainly to the rhizodermal cell layer. This is particularly true for roots growing in soil (see Section 2.10). More recently, also in solution culture the particular role of the rhizodermal cells for uptake of nitrate (Deane-Drummond and Gates, 1987) and sulfate (Holobrada and Kubica, 1988) has been demonstrated and also the predominant location of plasma membrane-bound H"̂ pumping ATPases in the rhizodermal cells of roots (Felle, 1982; Parets-Soler et al,, 1990). Other examples for the key role of rhizodermal cells in ion uptake are given in Section 2.7. 34 Mineral Nutrition of Higher Plants 5 6 7 Solution pH Fig. 2.14 Relationship of solution pH, proportion of H2PO^ ( ), and uptake rate of phosphate ( ) and sulfate ( ) by bean plants; relative values. (After Hendrix, 1967.) 2.5.2.3 Metabolic Activity In order for ions and other solutes to accumulate against a concentration gradient, an expenditure of energy is required, either directly or indirectly. The main source of energy in nonphotosynthesizing cells and tissues (including roots) is respiration. Thus, all factors which affect respiration may also influence ion accumulation. The few examples below demonstrate this connection. Oxygen. As oxygen tension decreases, the uptake of ions such as potassium and phosphate falls, particularly at very low oxygen tensions (Table 2.13). Consequently, oxygen deficiency is one of the factors which may restrict plant growth in poorly aerated substrates (e.g., waterlogged soils; Section 16.4). Carbohydrates: The main energy substrate for respiration are carbohydrates. There- fore, in roots and other nonphotosynthesizing tissues, under conditions of limited carbohydrate supply from a source (e.g. leaves) a close correlation can often be found between carbohydrate content and the uptake of ions, e.g. potassium (Mengel, 1962). In roots within a few hours after excision with depletion of carbohydrate content Table 2.13 Effect of Oxygen Partial Pressure around Roots on Uptake of Potassium and Phosphate by Barley Plants'" Uptake^ Oxygen partial pressure (%) Potassium Phosphate 20 5 0.5 100 75 37 100 56 30 "Based on Hopkins et al. (1950). ''Data represent relative values. Ion Uptake Mechanisms of Individual Cells and Roots 35 Table 2.14 Effect of Root Excision on Sugar Content, Respiration (O2 Uptake), and Nitrogen Uptake in Barley Roots'" Time (h) after excision 0 3 Sugar content {jumolg~^ dry wt) 82 51 Net uptake (jumol g"^ dry wt min~^) O2 NH4+ NO3- 4.5 1.8 1.5 3.3 1.1 1.0 "Recalculated from Bloom and Caldwell (1988). respiration and nitrogen uptake also decrease (Table 2.14). These relationships are of particular ecological importance, for example, when leaves are removed (grazing, cutting) or in dense plant stands when hght supply to the basal leaves is limited, since basal leaves are the main source of carbohydrates for the roots. Root carbohydrate content may also become a growth-limiting factor at high supply of ammonium nitrogen in combination with high root zone temperatures (Kafkafi, 1990; see also Section 8.2.4). Diurnal fluctuations in net proton excretion (Vogt et al., 1987) and in ion uptake rate by roots may also be at least in part causally related to corresponding fluctuations in carbohydrate supply to the roots. Distinct diurnal patterns in uptake rates (maxima during the day, minima during the night) can be observed for nitrate (Clement et al., 1978b) and nitrate and potassium (Le Bot and Kirkby, 1992). Root carbohydrate content may act as a coarse control for ion uptake and is one of the factors responsible for the diurnal fluctuations in ion uptake. However, in maize roots, for example, diurnal fluctuations in nitrate uptake were only loosely related to the root carbohydrate content (Fig. 2.15). In contrast, the relations were close between root carbohydrate content and nitrate reductase activity (NRA). There is good evidence that the root carbohydrate content as a regulating factor for diurnal fluctuations in ion uptake is superimposed by internal factors such as nutrient demand or retranslocation of nutrients or both (Section 2.5.6). In soybean growing under short-day conditions, the typical diurnal fluctuations of nitrate uptake could be reversed by an intervening 3 h period of low hght (i.e. imitating long-day conditions, repressed flower initiation); uptake rates of nitrate were then twice as high during the night as compared with the day (Raper et a/., 1991). Temperature. Whereas physical processes such as exchange adsorption of cations in the AFS are only slightly affected by temperature (gio * ~ 1.1-1.2), chemical reactions are much more temperature dependent. An increase in temperature of 10°C usually enhances chemical reactions by a factor of 2 {QIQ ~ 2). For biochemical reactions, gio values considerably higher than 2 are quite often observed. Also, for the uptake of ions such as potassium, QIQ is often much higher than 2, at least within the physiological *Qio, or quotient 10 refers to the change in the rate of a reaction or process (i.e., rate of membrane transport) imposed by a change in the temperature of 10°C. 36 Mineral Nutrition of Higher Plants o 33 Si 4 j 0) to 5 0^ 12 18 Hours 0.6 0.4 0.2 0 ^^"^ •? 3 o 2 o l\J •——1 (Q — h i 0) I T E h l ~ j 1 1 z r-fr 0) c? -̂ (D Q. C a SD CO CD 0) Q Fig. 2.15 Diurnal fluctuations in nitrate uptake ( ), nitrate reductase activity and content of water soluble carbohydrates in maize roots. Nitrate uptake: relative values, uptake at the end of the light period = 100. (Based on Keltjens and Nijenstein, 1987; by courtesy of Marcel Dekker Inc.) temperature range (Fig. 2.16). A comparison of the (2io values for ion uptake and respiration reveals that ion uptake is more temperature dependent, especially below 10°C. This may possibly indicate that in chilling-sensitive plant species like maize, restricted ion uptake at low temperatures is primarily the result of low membrane fluidity and strongly depressed activity of the plasma membrane-bound proton pump (Kennedy and Gonsalves, 1988). At supraoptimal temperatures root respiration further increases whereas ion uptake declines (Fig. 2.16), indicating again that respiration and mem- brane transport of ions are not directly coupled. In studies on temperature effects on ion uptake two major problems arise: short-term 10 20 30 Temperature (°C) 40 Fig. 2.16 Effect of temperature on rates of respiration (•) and uptake of phosphorus (O) and potassium (D) (supply of 0.25 mM potassium and 0.25 mM phosphorus) by maize root segments. (After Bravo and Uribe, 1981.) Ion Uptake Mechanisms of Individual Cells and Roots 39 Table 2.16 Interaction between Uptake of N H / and K"̂ in Maize Roots'"'^ (NH4)2S04 (mM) 0 0.15 0.50 5.00 Contents Ammonium 6.9 7.3 17.1 29.4 in roots (jumol (NH^) 6.7 7.1 13.5 31.5 g ^ fresh wt) Potassium - K + +K+ 8.2 53.7 6.7 48.4 8.9 41.1 9.3 27.1 "'Based on Rufty et al, (1982a). ^Duration of the experiment: 8 h; +K indicates addition of 0.15 mM K^; calcium concentration constant at 0.15 mM. Table 2.17 Effect of K-" and Ca^^ on the Uptake of Labeled Mg^^ (^^Mg) by Barley Seedlings^ Roots Shoots Mg2+ Uptake (juoq Mg^-'CIO g)"^ fresh wt (8 h)"^) Mga2 MgCl2 + CaS04 MgCl2 + CaS04 + KCl 165 115 15 88 25 6.5 "Concentration of each cation: 0.25 meq 1 ^ Based on Schimansky (1981). et al., 1987) would then be merely a reflection of competition for negative charges within individual cells, or in the whole plant (Engels and Marschner, 1993) that is of cation-anion relationships (Section 2.5.4). Of the mineral nutrients that are taken up as cations, the binding strength is rather low of the highly hydrated Mg^^ at the exchange sites in the cell walls (Section 2.2.1) and presumably also at the binding sites at the plasma membrane. Other cations such as K"̂ and Câ "̂ therefore compete quite effectively with Mĝ "̂ and strongly depress the uptake rate of Mg^+ (Table 2.17). This strong competition is in agreement with observations of magnesium deficiency induced in crop plants by extensive application of potassium and calcium fertilizers. A particular effective competition on Mg^^ uptake is exerted by Mn^^ (Table 2.18). Inhibition of Mg'̂ "̂ uptake by far exceeds the 1:1 competition for specific binding sites at the plasma membrane of root cells. Presumably, Mn̂ "̂ not only competes much more effectively but also in some way blocks the binding or transport sites for Mĝ "̂ or both. In contrast, the uptake of K"̂ is only shghtly depressed by increasing Mn^^ concen- trations (Heenan and Campbell, 1981). Competition and limited selectivity of binding sites at the plasma membrane are also observed for anions. Some well-known examples are competition between sulfate and 40 Mineral Nutrition of Higher Plants Table 2.18 Effect of Increasing Manganese Concentrations in the Substrate on Uptake Rates of Manganese and Magnesium in Roots of Soybean Plants'̂ Nutrient Manganese Magnesium 1.8 0.5 121.8 Manganese supply (/uu) 90 3.1 81.1 275 4.8 20.2 "Data represent micromoles of nutrient taken up per gram of root dry weight. Based on Heenan and Campbell (1981). molybdate, sulfate and selenate, and phosphate and arsenate. Increasing sulfate concentrations in the rooting medium strongly depress molybdenum uptake which is of beneficial effect for plant growth and animal nutrition on soils with toxic levels of molybdenum (Pasricha et al., 1911 \ Chatterjee et al. 1992) but may become a critical factor on low molybdenum soils (Section 8.3). Antagonistic interactions between selenate and sulfate are quite distinct and of considerable practical importance in view of both, the selenium requirement of humans and animals, and the increasing concern on excessive selenate levels in certain soils (Tanji etal., 1986). Increasing sulfate levels very effectively decrease selenate uptake and selenium content in plants (Mikkelsen and Wan, 1990), irrespectively of the selenium tolerance of the plant species (Section 10.5). Arsenate and phosphate are taken up by the same transport system in both, lower and higher plants, leading to excessive uptake and toxicity of arsenate in plants growing on soils with high arsenate levels. In Holcus lanatus L. arsenate-tolerant and nontoler- ant genotypes exist, and in the tolerant genotypes the arsenate uptake is much lower (Meharg and Macnair, 1992). This low arsenate uptake is achieved by suppression of the phosphorus deficiency-induced high affinity uptake system (Section 2.5.6) in the tolerant plants. This is of advantage in respect to restriction of arsenate uptake and thus, arsenate tolerance. It might, however, have consequences for phosphorus nutrition, unless other mechanisms of phosphorus acquisition of more importance than the high affinity system come into play in plants acquiring phosphorus from the soil (Meharg and Macnair, 1992; see also Section 15.4). The examples of strong competition between K"̂ and Rb"^ and between anions as s o l " and SeOl" demonstrate that the selectivity of the binding sites in root plasma membranes is not a reflection of the role of a given mineral element in plant metabolism, but merely a reflection of the physicochemical similarities between ions that are plant nutrients (e.g. K"̂ and SOl") and ions which have no function in metabolism (e.g. Rb^ and SeOl"). Plants are thus unable to exclude unneeded ions from uptake. This aspect has important practical implications in, for example, the channehng of certain heavy metals into the food chain via their uptake by plants (Marschner, 1983). Another distinct type of anion competition occurs between chloride and nitrate. The chloride content in plants, particularly in roots, can be strongly depressed by increasing Concentation in nutrient solution cr 1 1 1 1 (mM) NO3- 0 0.2 1.0 5.0 Ion Uptake Mechanisms of Individual Cells and Roots 41 Table 2.19 Effects of Nitrate Concentrations in the Nutrient Solution on Chloride Contents in Roots and Shoots of Barley Plants'" Chloride content (jumol g ^ fresh wt) Roots Shoot 52 93 26 73 13 54 9 46 ''Based on Glass and Siddiqi (1985). nitrate concentrations (Table 2.19). This depression seems to be the result of both, negative feedback effects from nitrate stored in the vacuoles of root cells as well as chloride influx at the plasma membrane (Glass and Siddiqi, 1985). It is still a matter of controversy whether nitrate and chloride are transported by the same (Pope and Leigh, 1990) or different (Dhugga et al., 1988) carrier systems. The net influx of nitrate is decreased by chloride, and the chloride already accumulated in the vacuoles seems to be particularly effective in this respect (Cram, 1973). Competition between nitrate and chloride during uptake is of great importance for crop production. The competing effect of chloride can be used to decrease the nitrate content of such plant species as spinach which tend to accumulate large amounts of nitrate and to use it mainly as an osmoticum (Section 8.2). On the other hand, in saline soils the competing effect of chloride on nitrate uptake may impair nitrogen nutrition of the plants (Bernal et al., 1974). Under these conditions increasing nitrate supply can be an effective means to improve the nitrogen nutritional status of the plants and simultaneously prevent chloride toxicity in sensitive plant species (Section 16.6.3). A particular case of competition is that exerted by ammonium on nitrate uptake. In almost all cases external ammonium strongly suppresses net uptake of nitrate. In contrast, externally supphed nitrate generally has little or no effect on net uptake of ammonium (Breteler and Siegerist, 1984). Accordingly by supplying NH4NO3, am- monium is usually taken up very much preferentially than nitrate, and at higher external ammonium concentrations, uptake of nitrate is suppressed until the ammonium concentration is considerably lowered. In Norway spruce such a threshold value for ammonium depletion is about 100 //M NH^ (Marschner et al, 1991). In short-term experiments with barley external ammonium inhibited net influx of nitrate within 3 min, and on removing the external ammonium net influx of nitrate resumed within 3 min (Lee and Drew, 1989). Rapid ammonium influx into the cytoplasm and a decrease in transmembrane potential are considered as factors possibly involved in the rapid suppression of net nitrate influx (H"*'-NO^ symport), an explanation that would also account for the inhibitory effect of ammonium on cation uptake, for example potassium (Table 2.16). 44 Mineral Nutrition of Higher Plants Table 2.20 Effect of Ca^+ on the Rates of K"̂ and C r Uptake in Barley Roots. External pH 5.0 External solution (mM) 0.1 KCl 0.1KCl-hl .0CaSO4 K"' Influx 116 ± 3 137 ± 2 Uptake rate (^eq g' K"̂ Net uptake 117 ± 6 140 ± 7 • M r y w t ( 2 h ) - i ) c r Influx c r Net uptake 35 ± 1 34 + 4 53 ± 3 52 ± 4 Table 2.21 Effect of Ca^^ on the K-'/Na"' Selectivity of Roots Uptake rate (//eq g ^ fresh wt (4 h) )̂ Maize Sugar beet External solution NaCl + KCl (10 meq T ^ each) — Calcium + Calcium'' Na-' 9.0 5.9 K^ 11.0 15.0 Na"' H- K"' 20.0 20.9 Na-^ 18.8 15.4 K^ 8.3 10.7 Na-^ + K-' 27.1 26.1 '='0.5mMCaCl2. are supplied after a period of deprivation. In long-term experiments involving different growth rates, when 'concentration' or 'dilution' of mineral nutrients in the dry matter plays an important role, the interpretation of mutual effects of ions during uptake is rather difficult and should be undertaken with care. An example of synergism is Ca^"^-stimulated cation and anion uptake, first dis- covered by Viets (1944). As has been shown in Fig. 2.17 Câ "̂ stimulates net uptake of K"̂ at low pH, mainly by counteracting the negative effects of high H^ concentrations on plasma membrane integrity and functioning of the proton efflux pump. Accordingly at low external pH Câ "̂ not only enhances net influx of K"*" (countertransport) but also of anions as Cl~ (proton-anion cotransport). An example for this is given in Table 2.20 for 'low salt' barley roots (i.e. with low internal concentrations of K"̂ and CI"). Due to its stabilizing effect on the plasma membrane Câ "*" also plays an important role in selectivity of ion uptake, the K^/Na"^ selectivity of roots in particular (Table 2.21). Despite the genotypical differences in the K'̂ /Na"^ uptake ratio between the 'natrophobic' maize and the 'natrophilic' sugar beet (Section 10.2) in both plant species the presence of Câ "̂ shifts the uptake ratio in favour of K"̂ at the expense of Na'*'. In this experiment only net uptake rates were measured. Calcium may have exerted this influence on K^/Na"^ selectivity via stimulation of the Na"^ efflux pump, either as countertransport (antiport) of K'^/Na^ (Jeschke and Jambor, 1981) or of H'^/Na"^ (Mennen et aL, 1990), or via general effects on plasma membrane integrity, e.g. affecting ion channels. Calcium as divalent cation stabilizes biomembrane^ and favours a high transmem- Ion Uptake Mechanisms of Individual Cells and Roots 45 Table 2.22 Effect of the Accompanying Ion on the Rate of K"̂ and CP Uptake by Maize Plants^ Uptake rate (juoq g ^ fresh wt h )̂ K+ from C r from Concentration (meqr^) KCl K2SO4 KCl CaCla 0.2 2.0 20.0 1.6 2.7 5.7 1.6 1.9 2.2 0.8 2.0 4.3 0.7 1.0 2.1 "Recalculated from Liittge and Laties (1966). brane electropotential by reacting with negatively charged groups (e.g. of phospho- lipids) thereby influencing the physicochemical properties and functioning of biomem- branes. This function of Câ "̂ is reflected, for example, in higher efflux rates of low- molecular-weight solutes from calcium deficient roots and membrane vesicles from those roots (Matsumoto, 1988) as well as lower selectivity in ion uptake when it is absent in the external solution (Table 2.21). Calcium can be removed fairly readily from its binding sites at the outer surface of the plasma membrane, by chelators, for example (van Steveninck, 1965), or can be exchanged by high concentrations of H"̂ or metal cations including Na"^ (Lynch et al., 1987). Accordingly, for fulfilling its function in the plasma membrane of roots, the requirement of Câ "̂ in the external solution is dependent on these environmental factors. High Câ "*" concentrations are particularly required, for example, in saUne substrates, for maintenance of high K^/Na^ selectivity in uptake and for the salt tolerance of plants (Section 16.6). 2.5.4 Cation-Anion Relationships Because cation uptake and anion uptake are regulated differently (Fig. 2.9), direct interactions between cations and anions do not necessarily occur. For instance, at low external concentrations the uptake rate of a cation is not aiffected by the accompanying anion and vice versa, as shown in Table 2.22 for K"̂ and CI". At high external concentrations, however, an ion which is taken up relatively slowly can depress the uptake of an oppositely charged more mobile ion: for example, SOl" depresses K"̂ uptake, Câ ~̂ depresses Cl~ uptake. Different uptake rates of cations and anions require both regulation of cellular pH and compensation of electrical charges. Obviously, at high external concentrations this regulation becomes a limiting factor for the uptake of K"̂ when accompanied by SOl" and for Cl~ when accompanied by Câ "̂ (Table 2.22). Under these conditions, nonspecific competition between ions of the same charge can also occur. For example, cations such as K^ which are rapidly transported across the plasma membrane may depress the uptake rate of cations with slower transport rates such as Mg^^ and Ca^" ,̂ not by competition for binding sites at the plasma membrane, but by nonspecific 46 Mineral Nutrition of Higher Plants Table 2.23 Relationship between Cation-Anion Uptake and Organic Acid Content in Isolated Barley Roots'" External solution ( m e q r ^ ) 2 K2SO4 IKCl 1 CaCl2 Uptake {juQq g Cations 17 28 1 Afresh wt) Anions 1 29 15 Change in organic acid (jueq$~^ fresh wt) + 15.1 -0 .2 - 9 . 7 '^C02 Fixation (relative) 145 100 60 ''Based on Hiatt (1967a, b) and Hiatt and Hendricks (1967). competition for 'native' anions in the cytoplasm or the vacuole, if the rate of synthesis of these anions becomes limiting. In principle, different rates in the cation-anion uptake ratio by roots are an important cause for intracellular pH perturbations. The stabilization of cytosolic pH in the range 7.3-7.6 (Section 2.4.2) is achieved by the so-called pH stat which consists of two components, the biophysical pH stat, characterized by proton exchange through the plasma membrane or tonoplast (Fig. 2.9), and the biochemical pH stat which mainly involves production and consumption of protons, achieved by the formation and removal of carboxylic groups (Davies, 1986; Raven, 1986). The functioning of the biochemical pH stat is reflected in Table 2.23 in the net changes in organic acid content in roots in relation to the imbalance in cation-anion uptake ratio. When K2SO4 is suppUed, the excess cation uptake is compensated by an equivalent net synthesis of organic acid anions, and the excess of inorganic anion uptake with CaCl2 supply by a decrease in organic acid anions. These changes in carboxylation and decarboxylation of organic acids are also reflected in the different rates of CO2 fixation in the roots (dark fixation). The main reactions involved in the biochemical pH stat in relation to different cation- anion uptake ratios are shown schematically in Fig. 2.19. Excessive cation uptake (Fig. 2.19A) is correlated with pH increase in the cytoplasm and induces enhanced synthesis of organic acids, thereby providing anions (R • COO~) for pH stabilization and charge compensation and subsequent transport of cations and anions either into the vacuole or the shoot. In contrast, excessive anion uptake (Fig. 2.19B) is correlated with pH decrease in the cytoplasm (e.g. proton-anion cotransport. Fig. 2.9). Maintenance of high cytoplasmic pH requires enhanced decarboxylation of organic acids from the storage pool (i.e. the vacuoles). As consequences of this biochemical pH stat, imbalance in cation-anion uptake ratio increases or decreases root concentrations of organic acid anions and also the pH in the root apoplasm and external solution. In the experiment recorded in Table 2.23 when K2SO4 was supplied, the net efflux of H^ was 4.3 /^eq g~^ root fresh weight per 2 h, leading to a decrease in external pH from 5.60 to 5.12 (Hiatt and Hendricks, 1967). The cation-anion balance in plants and the conse- quences for rhizosphere pH and mineral nutrition of plants has been reviewed recently by Haynes (1990) and is discussed further in Section 15.3. In the cytoplasm the equihbrium between carboxylation (CO2 fixation) and decar- boxylation is regulated mainly by the pH sensitivity of two enzymes, PEP carboxylase Ion Uptake Mechanisms of Individual Cells and Roots 49 Table 2.24 Influence of the Form of Nitrogen Supply on the Ionic Balance in the Shoots of Castor Bean Plants^ Form of N supply NO3- NH4+ K-̂ 99 55 Cations Ca -̂̂ Mg2+ 85 28 43 22 Total 212 120 NO3- 44 0 H2PO4- 18 23 Anions SO|~ C r Organic acids^ 11 2 137 33 5 59 Total 212 120 ^Van Beusichem et al. (1988); data in meq (100 g)~^ dry wt. ^Calculated from the difference of Cations - Anions. 0 7 6 X 0. 5 4 3 V- T ^ 1 1 0 1 .—•- T-^ 2 " ^ ^ ^ T 3 4 Time (days^ NO3- - • — - • — • - — • " • NO3+NH/ / 1 1 1 '̂ 5 6 7 1 Fig. 2.21 Time course of external solution pH when sorghum plants were supplied with only NOf, only NH^, or both at a ratio of 8 NOf to 1NH^. Total nitrogen concentration, 300 mg 1" . (Redrawn from Clark, 1982b, by courtesy of Marcel Dekker.) affected whether nitrogen is supplied as NH^ or NOf (Fig. 2.21). In NO^-fed plants with preferential NO^ reduction in the roots such as sorghum, the external pH usually increases considerably with time, and with mixed supply, after preferential NH^ uptake and depletion of external NH^ the pH decrease is transient followed by pH increase as typically for NOf-fed plants. Interestingly, in NO^-fed plants the typical pH increase may also be reversed to a drastic pH decrease under conditions where NO J uptake and assimilation are impaired and cation-anion uptake ratio increased considerably. This situation is typical in many plant species for phosphorus deficiency (Schjorring, 1986), zinc deficiency (Cakmak and Marschner, 1990) and iron deficiency in dicots (Section 2.5.5). In NH4'-fed plants net proton excretion and thus pH stat in the roots becomes increasingly difficult at low external pH (Fig. 2.21). Particularly at high levels of NH4" supply, the pH in the root tissue in general (Findenegg et al, 1982) and also in the cytoplasm of root cells (Gerendas et al, 1990) may decrease substantially. Poor growth of NH^-fed plants at low external pH (Findenegg et al., 1982) is most probably related at least in part to the difficulty of pH stat regulation in the cytoplasm (Section 8.2). Maintenance of pH stat involves costs in terms of photosynthates and water (Raven, 50 Mineral Nutrition of Higher Plants External concentration Fig. 2.22 Schematic presentation of uptake rates of potassium, sodium, and boron in barley roots with increasing supply of KCl + NaCl and boron. Uptake rates of other mineral elements in brackets. 1985). This is particularly true in relation to nitrogen nutrition. When both NH^ and NO^ are supplied, pH stat may be achieved by similar rates of H"̂ production (NH4̂ assimilation) and H"̂ consumption (NO^ assimilation) and thus have a very low energy requirement (Raven, 1985; Allen et al., 1988). This may explain at least in part that optimal growth for most plant species is usually obtained with mixed supply of NH^ and NO3-(Section 8.2.4). 2.5.5 External Concentrations As discussed in Section 2.4.3 the uptake rate of ions such as K"̂ is usually governed in the low concentration range by saturation kinetics. It is also highly selective and closely coupled to metabolism. In contrast at high external concentrations the uptake rate is more or less linear, is not very selective, not particularly sensitive to metabolic inhibitors (Glass et aL, 1990) and probably reflects ion transport through channels. In Fig. 2.22 the typical relationships between external concentration and uptake rate of K"̂ are presented schematically, without consideration of Cmm (Section 2.4.3). In principle similar uptake isotherms have been obtained for phosphorus (Loneragan and Asher, 1967), nitrate (Glass etal., 1990) and sulfate (Clarkson and Saker, 1989). In contrast to K^ the uptake rate, for example, of Na"̂ is much more concentration dependent, reflecting less specific binding sites in the plasma membrane - or a higher efficiency of a K^-Na^ coupled Na"̂ efflux pump. A similar uptake isotherm as for Na^ is often found for Mg^^ and Câ "̂ . The question arises as to the ecological importance of these differences in uptake isotherms. Compared with the requirement for optimal growth the concentrations in soil solutions are usually low for K^ ( « 1 mM) and phosphate (<0.05 mM); on the other hand, the concentrations of Câ "̂ and Mĝ "̂ are often considerably higher (Section 13.2). To satisfy the different requirements for these nutrients, plants have binding sites in the plasma membrane of root cells which differ in affinity (^m) and probably in number for the various mineral nutrients. As already stressed, however, in Section 2.5 the uptake isotherms and their parameters {K^; /max) for a given nutrient can also be strongly modulated by environ- mental (e.g., temperature) and internal factors (e.g., nutritional status). Another Ion Uptake Mechanisms of Individual Cells and Roots 51 Table 2.25 Influx of Nitrate in Barley Roots without and with Induced High Capacity Nitrate Uptake System'̂ External cone. NOf influx (^mol g"^ fresh wt h~^) (mM NOf) non-induced induced 1 day^ induced 4 days^ 0.02 0.10 2.75 1.54 0.30 0.39 5.27 2.86 20.0 11.54 20.87 8.02 ''Based on Glass et al. (1990). ^Pretreatment with nitrate for one or four days. Table 2.26 Effect of Increasing Boron Supply on Boron Content in Shoots and Shoot Dry Weight of Two Barley Genotypes" Boron supply (juu) Boron content (mg kg"^ dry wt) Schooner Sahara 3771 Shoot dry weight mg per plant Schooner Sahara 3771 0 5.6 2.5 129 74 2.5 10.0 5.5 140 84 7.5 22.1 7.8 132 92 15 46.4 11.7 121 107 "Based on Nable et al. (1990a). modulating factor is the induction of an uptake system by external supply of this nutrient. In plants grown in the absence of nitrate (noninduced) roots possess only a low capacity (constitutive) uptake system for nitrate. After nitrate supply, however, within 20 min a high capacity uptake system is formed for which protein synthesis is required (Mack and Tischner, 1990). An example showing this is given in Table 2.25 for barley roots. The uptake capacity of the non-induced system is very low and with increasing external nitrate concentrations the uptake isotherm resembles features similar to that of Na"*" (Fig. 2.22), i.e. a large contribution of a passive, concentration-dependent response with a low response to metabolic inhibitors (Glass et al., 1990). In contrast, in the induced roots (Table 2.25) the affinity and the uptake capacity for nitrate are much higher and the uptake isotherm resembles that shown in Fig. 2.22 for potassium and phosphate. Interestingly, after 4 days' induction the influx rate of nitrate is much lower, indicating a feedback regulation of the internal concentration (Section 2.5.6). Compared with other mineral nutrients the isotherm of boron is unique. In both individual cells (Seresinhe and Oertli, 1991) and plants (Fig. 2.22) the uptake rate of boron is linearly related to the external concentration (Table 2.26). Despite this linear relationship there are distinct genotypical differences in total boron uptake. The 54 Mineral Nutrition of Higher Plants > < • « o 9 a> "o E c 1400 1200 1000 800 600 400 200 0 A / 1 i tf'' — 1 1- ,5--' - ^ ' 1 so/- Pi —t 1 1- "̂1 i — — ) — L \ ^ in- so/-" -•s 1 2 3 4 Time (days) 0 10 20 Time (hours) Fig. 2.24 Time course of the influx of sulfate (SO4") and phosphate (Pj) in roots of barley plants deprived of external sulfate supply for up to 5 days (A) and then resupply of sulfate for up to one day (B). (Redrawn from Clarkson and Saker, 1989.) increased, either by increase in the turnover rate or number of the binding sites in the plasma membrane. Support for the latter mechanism is the increase in a 25 kDa polypeptide in the plasma membrane of roots deprived of phosphorus (Hawkesford and Belcher, 1991). The dynamics of this feedback regulation of the internal concentration on the uptake rate is shown in Fig. 2.24 for sulfate. Without external sulfate supply the capacity of barley roots for sulfate uptake (influx) increased rapidly within 3-5 days, but decreased drastically within a few hours and was lost within one day when sulfate was resupplied. In contrast, the influx rate of phosphorus was not affected by the sulfur nutritional status. Indications for an increase in number of specific binding sites for sulfate at the plasma membrane of root cells of sulfur deficient plants had been found in wheat (Persson, 1969). Induction of the enhanced transport system for sulfate requires protein synthesis and sulfhydryl groups seem to be involved in this induced system (Clarkson and Saker, 1989). In roots deprived of sulfate supply a 36 kDa polypeptide increases in the plasma membrane which might be a component of the plasma membrane sulfate transport system (sulfate permease; Hawkesford and Belcher, 1991). The mechanism, or the chemical nature, of the negative feedback signal on sulfate uptake might be the sulfate stored in the vacuoles (Cram, 1983), i.e. mechanism (3) in Fig. 2.23, or reduced sulfur compounds such as the amino acids cysteine and methionine (Brunold and Schmidt, 1978) or reduced glutathione (Rennenberg et al., 1989). The uptake rate of nitrogen is also closely related to the nitrogen nutritional status of the plants. For example, the uptake rate of NH4̂ is negatively related with the root concentrations of NH4^ and amino acids, glutamine and asparagine in particular (Causin and Barneix, 1993). Accordingly, the uptake rate of NH^ rises rapidly within a few days after withdrawal of nitrogen supply (Lee and Rudge, 1986). A decrease in NH4' efflux is involved but this is not the major factor responsible for the increase in NH^ uptake in N-starved plants (Morgan and Jackson, 1988). For nitrate uptake the mechanism of feedback regulation is more complex because of the apparently opposing effects of NO^, namely induction of a high capacity uptake system (Table 2.25) and Ion Uptake Mechanisms of Individual Cells and Roots 55 0 24 48 Duration of NO3" 72 96 120 144 pretreatment (hours) Fig. 2.25 Effect of NO:̂ -pretreatment on nitrate content (•) and NO^ influx (O) in roots of barley plants. (Siddiqi et al, 1989; reprinted by permission of the American Society of Plant Physiologists.) negative feedback regulation by increasing internal concentrations (Fig. 2.25), In noninduced plants, NO^ supply increases rapidly both the influx and the content of NOi" in the roots. With further increase in root content, however, the influx rate is depressed. This negative feedback regulation may be caused by high NO^ concen- trations in the vacuoles [Fig. 2.23 (3)] and expression of a turgor-regulated event (Cram, 1973; Glass, 1983). However, negative feedback regulation by elevated levels of reduced nitrogen in the form of the amino acids glutamine and asparagine (Lee et al,, 1992) or of NH^ is more likely [Fig. 2.23 (4)] as it is known from the inhibitory effects of NH^ supply on NO^ uptake (Section 2.5.4), or from reduced sulfur compounds on sulfate uptake (see above). Of course, transformation of a mineral nutrient in the root cells can also act as a positive feedback signal, for example, if combined with incorporation into macromolecular structures (proteins, nucleic acids) in growing cells. Similar to the examples shown for sulfate and nitrate, after phosphorus supply is withheld from the external solution, the uptake (influx) rate of phosphorus increases after resupplying phosphorus. However, those enhanced influx rates are transient and may last only for a few hours (Lefebvre and Glass, 1982). In this feedback regulation the internal concentration of inorganic phosphorus (Pi) plays a key role, in root cells probably its concentration in the vacuoles, rather than in the cytoplasm (Lee and Ratcliffe, 1983) where the Pj concentration is usually kept quite constant (Lee and Ratcliffe, 1993). In agreement with this, in intact plants the feedback regulation of elevated internal levels can be delayed for several days, because rapid phosphorus translocation into the shoot [Fig. 2.23 (5)] prevents a marked increase in phosphorus content within the roots (Table 2.28). Resupplying phosphorus after a period of deficiency can therefore lead to greatly increased phosphorus content in the shoots and also to phosphorus toxicity (Green and Warder, 1973). Such rapid changes in phos- phorus supply are unlikely to occur in soil-grown plants. In nutrient solution culture, however, this factor has to be considered, especially after replacement of solutions. Although these deficiency-induced new binding sites are specific for each nutrient they also have a limited selectivity in terms of functional requirement of a mineral element as plant nutrient. For example in barley, in sulfur-deficient plants selenate 56 Mineral Nutrition of Higher Plants Table 2.28 Contents of Phosphorus in Barley Plants after Growth without Phosphorus or Resupply of Phosphorus^ Phosphorus content (jumol P g~^ dry wt)^ 7 days - P 7 days - P 8 days - F + 1 day + P'' + 3 days + P^ Shoot total 49 (20) 151 (61) 412 (176) Youngest leaves 26 (5) 684 (141) 1647 (493) Roots 43(24) 86 (48) 169 (94) "Based on Clarkson and Scattergood (1982). ^Numerals in parentheses are relative values; 100 represents control with continuous phosphorus supply of 150/iM P throughout the experiment. '^ight days of growth without phosphorus. ''Seven days of growth without phosphorus and 1 day of growth upon addition of phosphorus (150 ̂ M). ^Seven days of growth without phosphorus and 3 days of growth upon addition of phosphorus (150 JUM). Uptake rate is also increased, and in phosphorus-deficient plants the uptake rate of arsenate is enhanced (Lee, 1982). Unusual and unexpected responses also sometimes occur. In tomato roots, for example, cadmium uptake rate increases with increase in cadmium content of the roots (Petit et al., 1978). This might reflect the induction of synthesis of compounds such as metallothioneins, or phytochelatins, which have a specific binding affinity for heavy metals (Section 8.3). A similar mechanism might be involved in the surprising differences in the rate of copper uptake in copper-sufficient and copper-deficient plants: on resupplying copper to deficient plants the uptake rate is much lower than in copper-sufficient plants (Jarvis and Robson, 1982). The relationships between influx rate and internal coiicentration of a particular mineral nutrient cannot always be explained satisfactorily by consideration of the roots alone. Positive and negative feedback control by the shoot [Fig. 2.23 (6)] can markedly affect the uptake rate of the roots. An example for this has been given for nitrate and potassium in Table 2.15. Such a feedback control is essejitial for the coordination of nutrient uptake depending on the demand of the plants for growth. A varied shoot supply of sugars to the roots (Pitman, 1972b), or different rates of root export of mineral nutrients in the xylem into the shoot [Fig. 2.23 (5)] can be considered as a coarse feedback control. However, there are also fine controls specific for particular nutrients. For example, the uptake rates of phosphorus (Drew et al., 1984) or potassium (Table 2.29) may be more closely related to the concentrations,of these nutrients in the shoots than in the roots. There is good evidence that retranslocation of mineral nutrients from shoot to roots may play an important role as a feedback.regulation signal from the shoot for ion uptake by the roots depending on shoot demand. This is the case for potassium and phosphorus as well as iron (Maas et al., 1988), hitrogen in form of amino acids (Simpson etal., 1982), and sulfur in the form of glutathione (Rennenberg, 1989). In tomato plants about 20% of the K^ flux in the xylem was cycling, i.e., originated from the shoots (Armstrong and Kirkby, 1979a), and in young wheat and rye plants the Ion Uptake Mechanisms of Individual Cells and Roots 59 Rhizoplane (Rhizosphere) Chelator ^ Apoplasnri Plasma Cytoplasm (Rhizodermis) membrane Fig. 2.27 Model for root responses to iron deficiency in dicots and non-graminaceous monocots; Strategy I: (R) inducible reductase; [TR] transporter or channel for Fe(II)?; (1) stimulated proton efflux pump; (2) increased release of reductants/chelators. (Modified from Marschner et aL, 1986a; Romheld, 1987a,b.) Table 2.30 Effect of Iron Deficiency in Cucumber (Strategy I) on Proton Excretion (pH), Reducing Capacity of the Roots and Iron Uptake Rate" Fe nutritional status (preculture) Chlorophyll (mg g~^ dry wt) H"̂ excretion (pH solution) Reducing cap. (jumol Fe(II) g"_̂ rootdrywt (4h)~^) Fe uptake (jumol g~^ root dry wt (4 h)-^) +Fe^ -Fe 12.2 7.8 6.2 4.8 3.2 96.8 0.03 2.6 ''Compiled data from Romheld and Kramer (1983) and Romheld and Marschner (1990). ^Supply of 1 X 10"^ M FeEDDHA, pH 6.2. Although the transmembrane redox pump may contribute to the net excretion of protons (Fig. 2.9) the strongly enhanced net excretion of protons under iron deficiency is most probably the result of higher activity of the plasma membrane proton efflux pump [Fig. 2.27 (1)] and not of the reductase (Bienfait et aL, 1989; Alcantara et aL, 1991). The activity of the reductase (R) which is strongly stimulated by low pH, i.e., enhanced proton excretion by the ATPase is important for the efficiency in Fe(III) reduction. Accordingly, high concentrations of HCOi" counteract this response system in Strategy I plants (Section 16.5.3). An example for root responses to iron deficiency and the corresponding enhanced uptake rates of iron are shown in Table 2.30 for cucumber as a Strategy I plant. The much higher reduction rates of Fe(III) at the outer surface of the plasma membrane of root rhizodermal cells are correlated with distinctly enhanced uptake rates of iron. It is not clear whether the reductase itself or an attached protein (Fig. 2.27 [TR]) mediates 60 Mineral Nutrition of Higher Plants the transport of the reduced iron [Fe(II)] into the cytoplasm (Young and Terry, 1982; Grusak et al., 1990). It is also not clear how the iron nutritional status of the cells is transformed into a 'signal' for the response mechanisms at the plasma membrane (Bienfait, 1989). The involvement of nicotianamine as chelator for Fe(II) and a sensor for iron nutritional status has recently been discussed (Pich and Scholz, 1991). Both root responses, increase in reduction rates of Fe(III) and iron uptake, are under strict genetic control (Alcantara etal., 1990; Romera etal., 1991) and, in a given plant species (chickpea) the responses seem to be controlled by only one dominant gene (Saxena et fl/.,1990). Strategy / / i s confined to graminaceous plant species (grasses) and characterized by an iron deficiency-induced enhanced release of non-proteinogenic amino acids, so-called phytosiderophores (Takagi etal., 1984). The release follows a distinct diurnal rhythm and is rapidly depressed by resupply of iron (Fig. 2.28). The diurnal rhythm in release of phytosiderophores in iron-deficient plants is inversely related with the volume of a particular type of vesicles in the cytoplasm of root cortical cells (Nishizawa and Mori, 1987). Phytosiderophores such as mugineic acid (Fig. 2.29) form highly stable complexes with Fe(III), the stability constant in water is in the order of 10^^ (Murakami et al., 1989). As a second component of Stragegy II a highly specific constitutive transport system (Translocator (Tr), Fig. 2.29) is present in the plasma membrane of root cells of grasses (Romheld and Marschner, 1990) which transfers the Fe(III) phytosiderophores into the cytoplasm. In plant species with Strategy I this transport system is also lacking. Although phytosiderophores form complexes also with other heavy metals such as zinc, copper and manganese (Fig. 2.29), the translocator in the plasma membrane has only a low affinity to the corresponding complexes (Marschner et al., 1989; Ma et al., 1993). Nevertheless, release of phytosiderophores may indirectly enhance the uptake rate of these other metals by increasing their mobility in the rhizosphere and in the root apoplasm {Zhangetal., 1991a,b,c). Under iron deficiency not only the release of phytosiderophores is increased but also 0) E g 11 12 13 14 Age of plants (days) 0 6 12 18 Time of day Fig. 2.28 Release of phytosiderophores (PS) from barley roots as affected by the iron nutritional status (A), and diurnal rhythm in release of phytosiderophores (B). Fe sufficient (•) and Fe deficient (O) plants. (Based on Romheld, 1987a,b and A. Walter, personal communi- cation.) Ion Uptake Mechanisms of Individual Cells and Roots 61 COOH COOH OH Muqineic acid (MA) CH, "^^i " ^ ^ NFT" Rhizoplane (rhizosphere) Apoplasm IPM. Cytoplasm Phytosiderophores Fe"^PS Zn^PSCi/PS Mn^PS w 1—Fe^PS Zn'Cu" Fig. 2.29 Model for root responses to iron deficiency in graminaceous species; Strategy II: (E) enhanced synthesis and release of phytosiderophores; (Tr) translocator for Fe(III) phytosidero- phores in the plasma membrane; structure of the phytosiderophore mugineic acid and its corresponding Fe(III) chelate (Modified from Nomoto et al. 1987; Romheld, 1987b.) the uptake rate of the Fe(III) phytosiderophore complexes (Table 2.31) indicating a higher transport capacity (/max) due either to an increase in number or the turnover rate of the translocator. As mechanism of phytosiderophore release a cotransport with either protons or potassium is discussed, and for the uptake of Fe(III) phytosidero- phores a proton-anion cotransport across the plasma membrane (Mori etal., 1991). Although this phytosiderophore system resembles features of the siderophore system in microorganisms (Winkelmann, 1986) its affinity to phytosiderophores is two to three orders of magnitude higher than for siderophores such as ferrioxamine B (Bar-Ness et al., 1991,1992; Crowley et al,, 1992), or for synthetic iron chelates such as FeEDDHA (Romheld and Marschner, 1990; Bar-Ness etal., 1991, 1992). For the biosynthesis of phytosiderophores, methionine is the common precursor and nicotianamine an intermediate (Shojima et al,, 1990): Avenic acid Methionine -^ Nicotianamine -^ 2'-Deoxymugineic acid -> Mugineic acid -^ 3-Hydroxymugineic acid This pathway is under strict genetic control and the chromosomes have already been Table 2.31 Release of Phytosiderophores (PS; mugineic acid) and Uptake of Iron-Phytosiderophores in Iron-Sufficient and Iron-Deficient Barley Plants'' Fe nutritional Chlorophyll PS release Fe uptake status content (/̂ mol g"^ root (jumol g~^ root (preculture) (mg g~ ̂ dry wt) dry wt (4h) ~ ̂ ) dry wt (4h)" ̂ ) + Fe - F e 12.8 7.5 0.4 8.2 0.4 3.4 "Romheld and Marschner (1990). Reprinted by permission of Kluwer Academic Publishers. 64 Mineral Nutrition of Higher Plants Table 2.33 Effect of Phosphorus Nutritional Status on the Rate of Phosphorus Uptake by Various Root Zones of Barley Plants'̂ Pretreatment for 9 days With phosphorus Without phosphorus Root zone (distance from root tip, cm) 1 2 3 2019 1558 970 3150 4500 4613 '̂ Uptake rate expressed as pmoi mm ^ of root segment in 24 h. Based on Clarkson et al. (1978). the apical zone, despite the high potassium demand for growth in the apical root zones. The high potassium concentration in root apical meristematic cells of about 200 mM (Huang and Van Steveninck, 1989a) is obviously maintained not only by uptake from the external solution but also delivery via the phloem either from basal root zones (Table 2.32) or from the shoot (Section 2.5.6). In perennial plant species like Norway spruce the uptake rates of potassium are also lower in apical than basal zones of nonmycorrhizal longroots (Haussling etal, 1988). In contrast to potassium the uptake of magnesium and particularly calcium is much higher in apical than in basal root zones (Ferguson and Clarkson, 1976; Haussling etal., 1988). This is also shown in Table 2.32. Because of lack of phloem mobility root tips have to meet their calcium demand for growth by direct uptake from the external solution. Root apical zones take up high amounts of calcium not only for their own demand but also for delivery to the shoot (Table 2.32; Clarkson, 1984). The calcium uptake in basal root zones is usually low but may increase in basal zones where lateral roots are formed and penetrate the cortex (Haussling et al, 1988). The decline in phosphorus uptake along the root axis is much less than for calcium (Ferguson and Clarkson, 1975). In soil-grown maize this decline is mainly related to a decrease in root hair viability and, thus, in absorbing root surface area (Ernst et aL, 1989). The gradient in phosphorus uptake along the root axis also depends on the phosphorus nutritional status of the plant and may be reversed under deficiency in favour of the basal zones (Table 2.33). This is probably related to lower internal phosphorus concentrations in the basal zones which deliver phosphorus to apical zones under deficiency conditions. The situation is different under iron deficiency in plants with Strategy I (Section 2.5.6) where the apical, but not the basal, root zones increase their capacity for iron uptake by a factor of up to 100 (Romheld and Marschner, 1981b). In contrast to dicotyledonous plants and perennial plants, in graminaceous species like wheat and barley the rhizodermis and cortex cells of basal (older) regions of the roots collapse and die. There are also reports (Lascaris and Deacon, 1991a,b) of early progressive senescence of rhizodermis and cortex cells behind the root hair zone in otherwise healthy looking roots ('root cortical death', RCD). This would be detrimen- tal for the uptake of nutrients and water through the older root zones. However, the Ion Uptake Mechanisms of Individual Cells and Roots 65 Lateral roots Root hairs Muoilage Fig. 2.30 Schematic representation of anatomical changes along the axis of a maize nodal root. In basal zones there is degeneration of cortical cells and formation of tertiary endodermis. methods used for characterizing RCD had been questioned by Wenzel and McCully (1991). Formation of cortical gas spaces {aerenchyma) particularly in more basal root zones can often be observed (Fig. 2.30). Aerenchyma formation is a typical response to oxygen deficiency in plant species adapted to wetland conditions (Section 16.4). However, it can also be induced, for example, in maize roots under fully aerobic conditions by temporary deprivation of nitrogen or phosphorus supply (He et al., 1992). Despite these anatomical changes (Fig. 2.30) the basal root zones still have a consider- able capacity for ion uptake (Drew and Fourcy, 1986) and also for radial transport, indicating that the strands of cells bridging the cortex maintain sufficient ion transport capacity from the rhizodermis up to the endodermis (Drew and Saker, 1986). Gradients in uptake of water along the root axis may affect the gradients in ion uptake either indirectly via solute supply to the root surface (Section 13.2) or via direct effects on radial transport in the cortex. Water uptake rates are usually higher in the apical zones and decline thereafter sharply (Sanderson, 1983; Haussling et al, 1988). This decline in basal zones is caused mainly by formation of suberin in the rhizodermis, of the exodermis and the secondary and tertiary endodermis as efficient barriers against apoplasmic solute flow. In perennial species, in particular, water uptake may increase again in basal zones where lateral roots penetrate the cortex and temporarily disrupt these barriers (Haussling et al, 1988; MacFall et al, 1991). 2.7 Radial Transport of Ions and Water Across the Root There are two parallel pathways or movement of ions (solutes) and water across the cortex towards the stele: one passing through the apoplasm (cell walls and intercellular 66 Mineral Nutrition of Higher Plants Cortex Early metaxylenn Late metaxylem Phloem Endodermis Casparian band Exodermis (hypodermis) Rhizodermis Fig. 2.31 Part of transsection of a maize root showing the symplasmic (A) and apoplasmic (B) pathway of ion transport across the root. spaces, Section 2.2.1) and another passing from cell to cell in the symplasm through the plasmodesmata. In the symplasmic pathway ions, but not water, bypass the vacuoles (Fig. 2.31). As a rule the apoplasmic pathway of ions is constrained by the Casparian band in the walls of the endodermal cells. This band has hydrophobic properties and completely surrounds each cell. Additionally in basal root zones the cell walls of the endodermis become thicker and even lignified (tertiary endodermis). Recently, the key role of the endodermis and the Casparian band as effective barrier also for radial movement of water has been questioned. At least in young roots, despite the hydrophobic properties of the Casparian band, water seems to permeate this band readily (Peterson etal, 1993; Schreiber etal, 1994). Depending on the plant species and the root zone the apoplasmic pathway may already be constrained or blocked by the exodermis (Fig. 2.31) or suberization of the rhizodermis. Formation of an exodermis is found, for example, in Zea mays. Allium cepa, or Helianthus annum, but not in Vicia faba or Pisum sativum (Enstone and Peterson, 1992). However, there are somewhat different views on the function of the exodermis as effective barrier for transport of water and solutes in the apoplasm of the root cortex (Section 2.5.1). Termination of the apoplasmic pathway at the exodermis as suggested by Enstone and Peterson (1992) would confine in basal root zones both water influx and ion transport across the plasma membrane into the symplasm to the rhizodermal cells. Although rhizodermal cells play a key role in mineral nutrient uptake in general (Grunwald et al., 1979) and at low external concentrations of potassium and phosphate in particular (Drew and Saker, 1986), it is not possible to generalize on the relative importance of the two pathways in ion and water transport in the root cortex. This depends on: (a) the external concentration compared with the capacity and affinity of the transport system at the plasma membrane for a given ion (e.g., K^ > Na^; NOiT > H3BO3; Section 2.5.5); (b) the root zone considered: depending on the growth rate of the root, the exodermis may develop between 2 cm and 12 cm proximal to the root tip (Perumalla and Peterson, 1986) and may possess 'passage cells' (Storey and Walker, Ion Uptake Mechanisms of Individual Cells and Roots 69 c "o E -I—• c Q) *^ C o o 12 10 8 6 4 2 0 A > » < ^ ' y ^ - P' ____̂ ^o Shoots ,^' -*—• Roots __^_. : B / - < ^ " ^ - ' ^ ' ^^^ Roots p Shoots — i h — _ — 1 — — 2 4 6 8 1012 Tlnne (hours) 2 4 6 8 1012 Time (hours) Fig. 2.33 Accumulation and translocation rate of K"̂ Ĉ K̂) from 1 mM KCl (+0.5 mM CaS04) in barley plants (A) after preculture with 1 mM KCl or (B) without KCl. The mechanism of symplasmic transport of solutes seems to be chiefly diffusional, facilitated by radial water flux. Inhibition of cytoplasmic streaming may have no effect on the radial transport of potassium (Glass and Dunlop, 1979). On the other hand, in radial transport of phosphate, various metabolic steps such as incorporation into organic compounds (ATP, sugar phosphates) are involved before it is released as inorganic phosphate into the xylem (Sasaki et al,, 1987). During radial transport of ions competition occurs between accumulation in the vacuoles and transport in the sym- plasm. This competition depends on the mineral nutrient and its concentration in the vacuoles of root cells along the pathway. At low internal concentrations ('low-salt' roots), in short-term experiments this competition is reflected by a typical high accumulation rate in the roots and a delay in the translocation of the ions from the roots to the shoots of plants which originally were low in internal concentrations of the ion being investigated (Fig. 2.33). As a result of this competition, when the supply of a mineral nutrient is suboptimal the roots usually have higher contents of the particular nutrient than the shoot ('restricted translocation'). In long-term studies, this regulation mechanism is in part responsible for the often observed shift in the relative growth rates of roots and shoots in favor of the roots (Section 14.7). Along the symplasmic pathway preferential accumulation of certain ions like Na"^ may take place, accounting to some extent, for example, to the restricted shoot translocation of Na"^ in natrophobic plant species (Chapter 3). Preferential accumu- lation in roots is also a mechanism by which certain genotypes of maize ('shoot excluder') strongly restrict translocation of cadmium to the shoots (Florijn and Van Beusichem, 1993). On the other hand, symplasmic transport of phosphate and trans- location into the shoots is enhanced in expense of accumulation in vacuoles of the roots not only at high vacuolar concentrations of phosphate (see above) but also of nitrate (Lamaze et al., 1987). The exchange rate between ions in the vacuoles of cortex cells and those in the symplasm depends on the ion species (K"̂ > Na"^; NO^ > SOl") , the ty2 for exchange is in the order of at least a few days (Section 2.4.2). Radial transport of water and root hydraulic conductivity in general, and presumably also radial transport of ions, is strongly affected by maturation of xylem vessels along the root ^xis. For example, in graminaceous species like maize, growing in soil two types of roots are found; 'sheathed' with strong persisting soil sheath, and 'bare' roots 70 Mineral Nutrition of Higher Plants Soil sheath Nodal roots Fig. 2.34 Model of root hydraulic conductivity and formation of soil sheaths in the root system of maize and other C4 grasses. (Modified from Wenzel et al., 1989; reprinted by permission of the American Society of Plant Physiologists.) with no closely adhering soil (Fig. 2.34). This 'root dimoq^hism' is caused by differences in the maturation of the metaxylem (LMX) vessels (Fig. 2.31). In the sheathed zones the LMX vessels are still alive and nonconducting (McCulIy and Canny, 1988). Accordingly, the hydraulic conductivity of bare roots is about 100 times greater than that of sheathed roots (Wenzel et al., 1989). This difference leads directly to high moisture contents in the rhizosphere soil of the sheathed zones and low moisture of the bare zones (Fig. 2.34). Living LMX vessels persist up to 20-30 cm proximal to the root tip in maize (Wenzel et al., 1989), and up to 17 cm proximal to the root tip in soybean (Kevekordes et al., 1988). This delay in LMX maturation not only affects hydraulic conductivity of the roots and plant water relations (Wang et al., 1991) but also the mechanism of release of ions into the xylem and their transport to the shoot. 2.8 Release of Ions into the Xylem After radial transport in the symplasm into the stele, most of the ions and organic solutes (amino acids, organic acids) are released into the xylem. This release into fully differentiated non-living xylem vessels therefore represents a retransfer from the symplasm into the apoplasm. Crafts and Broyer (1938) postulated uphill transport in the symplasm across the cortex to the endodermal cells and, in the stele, a 'leakage' into the xylem. The distinctly lower oxygen tension in the stele than in the cortex of intact roots (Fiscus and Kramer, 1970) seemed to support this view of an oxygen deficiency- induced leakage. Also electrophysiological studies apparently indicate ion movement from the symplasm into the xylem along the electrochemical gradient (Bowling, 1981). In contrast, Pitman (1972a) put forward a two-pump model for ion transport from the external solution into the xylem, one located at the plasma membrane of root cortical cells and the other at the symplasm-xylem interface (apoplasm) in the stele (Fig. 2.35). Ion Uptake Mechanisms of Individual Cells and Roots 71 Fig. 2.35 Model for symplasmic (1) and apoplasmic (2) pathways of radial transport of ions across the root into the xylem. .Key:-0>, active transport; <—, resorption. (Modified from Lauchli, 1976a.) In this model the xylem parenchyma cells play a key role in ion secretion. High concentrations of ions such as K^ in these cells, together with transfer cell-like structures (Kramer et al., 1977) support this model. These cells seem to be involved also in reabsorption of ions from the xylem sap along the pathway to the shoot (Section 2.9). The release of ions and organic solutes {'xylem loading') is not well understood. The key role of a respiratory-dependent proton pump at the plasma membrane of the parenchyma cells is now well established. Protons are pumped into the xylem (DeBoer et al,, 1983; Mizuno et al,, 1985) and acidify the xylem sap which has pH values between about 5.2 and 6.0 depending, for example, on plant species and source of nitrogen supply (Arnozis and Findenegg, 1986). Similarly to the pump at the plasma membrane of cortical cells (Fig. 2.9) the proton pump at the plasma membrane of xylem parenchyma cells transfers protons into the apoplasm of the xylem vessels and may thereby act indirectly by reabsorption as a driving force for the secretion of cations (antiport). Anions may be secreted either by cotransport with the protons or along the electrical potential gradient formed by the proton pump (e.g. transport into the vacuole. Fig. 2.9). As the oxygen tension is usually lower in the stele than in the cortex, the xylem loading pump is more quickly inhibited at decreasing oxygen tensions in the root environment (DeBoer etal., 1983). Recently this concept of xylem loading as an energized process has been questioned by Wegner and Raschke (1994). By using isolated parenchyma cells from barley roots and measuring plasma membrane-potential related fluxes of cations and anions these authors suggest that similar to guard cells at closing (Section 8.7.6.2) also release of ions into the xylem sap occurs through ion channels in a process which is thermodynamically passive. Irrespective of the different views on the mechanism there is general agreement that xylem loading is separately regulated from the ion uptake in cortical cells. This separate regulation step offers the plant the possibility of control selectivity and rate of long- distance transport to the shoot, for example as a feedback regulation depending on shoot demand [Fig. 2.23 (6)]. For example, preferential xylem loading of nitrate 74 Mineral Nutrition of Higher Plants Table 2.36 Relationship between External Concentration, Exudate Concentration, and Exudate Volume Row in Decapitated Sunflower Plants External solution KNO3 + CaCl2 (niM each) Exudate (niM) 'Concentration factor' K^ Ca-2+ NO3 K+ Ca^ NO3 Exudation volume flow (ml(4h)- i ) 0.1 1.0 10.0 7.3 10.0 16.6 2.8 3.2 4.2 7.4 10.7 10.3 73 10 1.7 28 3.2 0.4 74 10.1 1.0 4.0 4.5 1.6 the rise in external concentration from 1.0 to 10.0 mM, does not compensate for the decrease in the exudation volume flow. Thus, in contrast to the accumulation in roots (hyperboHc function of the external concentration, see e.g., Fig. 2.11), the rate of root pressure-driven shoot transport of mineral nutrients can decline at high external concentrations. An increase in the root zone temperature has a much greater effect on the exudation volume flow than on the ion concentration in the exudate (Table 2.37). This is consistent with the expectation that a root behaves as an osmometer: Temperature determines the rate of ion transport in the symplasm (plasmodesmata, Section 2.7) and the release into the xylem, and water moves accordingly along the water potential gradient. There are, however, distinct differences between a root and a simple osmometer. A n increase in the root temperature results in an increase in the potassium concentration but a decrease in the calcium concentration of the exudate. This shift in the potassium/calcium ratio might reflect temperature effects either on membrane selectivity or on the relative importance of the apoplasmic pathway of radial transport of calcium and water (Engels et aL, 1992). Similar shifts in the potassium/calcium translocation ratio are also observed at different soil temperatures (Walker, 1969). This temperature effect may have important implications for the calcium nutrition of plants and might explain the enhancement of calcium deficiency symptoms in lettuce at elevated root temperatures, despite a slight increase in the calcium concentration of the leaf tissue (Collier and Tibbits, 1984). Table 2.37 Temperature Effect on Exudation Volume Flow and on Potassium and Calcium Concentration in the Exudate of Decapitated Maize Plants^ Temperature CO Exudation volume flow (inl(4h)->) Exudate concentration (mM) K+ Ca 2+ Ratio K-'/Ca^-' 8 18 28 5.3 21.9 31.7 13.4 15.2 19.6 1.5 1.0 0.8 8.9 15.2 24.5 ''Concentration of KNO3 and CaCl2 in the external solution: ImM each. Ion Uptake Mechanisms of Individual Cells and Roots 75 Table 2.38 Effect of Root Respiration on Exudation Volume Flow and Ion Concentration in the Exudate of Decapitated Maize Plants'^ Treatment^ O2 N2 Exudation volume flow (ml(3h)- 26.5 5.7 • ' ) Exudate centratioi] K+ 16.6 15.2 con- i(mM) Ca^^ 1.8 1.7 ''Concentration of KNO3 and CaCl2 in the external solution: 0.5 mM each. ^Respiration treatment consisted of bubbling oxygen or nitrogen through the external (nutrient) solution. The rate of release of ions into the xylem is closely related to root respiration (Table 2.38). A lack of oxygen strongly depresses the exudation volume flow but not the concentrations of potassium and calcium in the exudate. Oxygen deficiency seems to affect ion release into the xylem and root hydrauHc conductivity to the same degree. As in ion accumulation in root cells, maintenance of the cation-anion balance is necessary in the xylem exudate (Allen et al., 1988; Findenegg et al., 1989). Therefore, the accompanying ion may affect the transport rate even at low external concentrations (Table 2.39). When KNO3 is supplied, the exudation flow rate is almost twice as high as the flow rate when an equivalent concentration of K2SO4 is added. Since the potassium concentration in the exudate is similar in both treatments, the transport rate of potassium supplied as K2SO4 is only about half the rate of potassium supplied as KNO3. In contrast to the potassium concentration, the concentrations of nitrate and sulfate in the exudate exhibit a large difference (18.1 and 0.6 meq 1~\ respectively) between the treatments. The corresponding difference in negative charges in the exudate is approximately compensated for by elevated concentrations of organic acid anions. In the K2SO4 treatment, however, the rate-limiting factor is probably the capacity of the Table 2.39 Flow Rate and Ion Concentration in the Xylem Exudate of Wheat Seedlings'̂ Parameter Exudation flow rate {jul h~^ per 50 plants) Ion concentration (jucq ml"^) Potassium Calcium Nitrate Sulfate Organic acids Treatment KNO3 372 23.3 9.1 18.1 0.2 9.6 K2SO4 180 24.5 9.5 0.0 0.8 25.8 ''Seedlings were supplied with either KNO3 (1 mM) or K2SO4 (0.5 urn) in the presence of 0.2 mM CaS04. From Triplett et al. (1980). 76 Mineral Nutrition of Higiier Plants Table 2.40 Relationship between Photoperiod, Carbohydrate Content of Roots, and Uptake and Translocation of Potassium in Decapitated Maize Plants'^. Photoperiod (h) 12/12^ 24/0 Carbohydrate in roots (mg) 122 (48) 328 (226)^ Total potassium uptake (meq) 1.3 5.0 Potassium translocation in exudation volume flow (meq) 1.0 3.5 Exudation volume flow (ml (8 h)"^) 30.3 88.5 Relative decline in flow rate within 8 h (%) 60 12 ''Data per 12 plants. ^Hours of light/hours of darkness. This pre treatment with different day lengths was for one day (i.e., the day prior to decapitation). 'Numbers in parentheses denote carbohydrate content after 8 h (decapitation). roots to maintain the cation-anion balance by organic acid synthesis; this leads to a decrease in the rate of potassium and calcium release into the xylem and a correspond- ing decrease in exudation flow rate. Release of ions into the xylem and the corresponding changes in root pressure are also closely related to the carbohydrate status of the roots (Table 2.40). Variation in the length of the photoperiod for one day before decapitation affects the carbohydrate status of the roots and correspondingly the rate and duration of exudation volume flow after decapitation. Both the uptake and translocation rate of potassium in roots with a high carbohydrate content are considerably greater than in roots that are low in carbohydrate. The higher translocation rate is closely related to the exudation volume flow. In roots with a low carbohydrate content, reserves are rapidly depleted after decapitation and there is a corresponding decline in the rate of exudation volume flow within 8 h. This depletion of carbohydrates in the roots of decapitated plants is one of the factors which limits studies on exudation volume flow. However, release of ions into the xylem and exudation volume flow are not necessarily related to the carbohydrate status of the roots but can show endogenously regulated, distinct diurnal fluctuations which are also maintained in plants transferred to continuous darkness (Ferrario et al., 1992a,b). Hormonal effects may be involved in this endogenous rhythm as, for example, abscisic acid (ABA) strongly enhances exudation volume flow (Fournier et al, 1987). 2.9.2 Xylenn Exudate, Root Assimilation and Cycling of Nutrients Analyses of xylem exudates provide valuable information about assimilation of mineral nutrients in the roots, for example, or the capacity of roots for nitrate reduction or in legumes for N2 fixation. In soybean and other tropical legumes the proportion of ureides (Chapter 7) to total nitrogen in the xylem exudate reflects the nodule activity and is also a suitable indicator in field-grown plants of the relative contribution of N2 fixation to the nitrogen nutrition of legumes (Peoples et al, 1989). In non-legumes, analysis of the forms of nitrogen in the xylem sap can provide valuable information on Long-Distance Transport in the Xylem and Phloem and Its Regulation 3.1 General The long-distance transport of water and solutes - mineral elements and low-molecular- weight organic compounds - takes place in the vascular system of xylem and phloem. Long-distance transport from the roots to the shoots occurs predominantly in the nonliving xylem vessels. Coniferous trees lack the continuous system of xylem vessels, and depend on tracheides which are non-living conducting cells ranging in length from 2 to 6 mm (Tyree and Ewers, 1991). In annual plant species too long-distance transport in the xylem vessels may be interrupted by tracheides, for example at the root-shoot junction (Aloni and Griffith, 1991) or in the nodes of the stem. These structures pose an internal resistance to xylem volume flow but simultaneously permit an intensive xylem- phloem solute transfer (Section 3.3.4). Xylem transport is driven by the gradient in hydrostatic pressure (root pressure) and by the gradient in the water potential. As a reference the water potential of pure free water is defined as having a water potential of zero. Accordingly, values for water potential are usually negative. The gradient in water potential between roots and shoots is quite steep particularly during the day when the stomata are open. Values become less negative in the following sequence: atmosphere » leaf cells > xylem sap > root cells > external solution. Solute flow in the xylem from the roots to the shoots is therefore unidirectional (Fig. 3.1). However, under certain conditions in the shoots a Fig. 3.1 Direction of long-distance transport of mineral elements in non- living xylem vessels and in the phloem in roots. -f' • r h 80 Mineral Nutrition of Higher Plants Table 3.1 Accumulation and Long-Distance Transport of "̂ ^Ca, ̂ ^Na and "̂ K̂ in Maize Seedlings'"'^ Plant part Shoot Endosperm 24-27 cm root 21-24 cm root 18-21 cm root 15-18 cm root 12-15 cm zone of supply 9-12 cm root 6- 9 cm root 3 - 6 cm root 0- 3 cm root Total Content (jucq per 12 plants (24 h) )̂ ^^Ca 2.20 0.18 0.01 0.01 0.01 0.01 0.40 0 0 0 0 2.82 ^^Na ^^K 0.01 9.07 0.04 2.38 0.06 0.35 0.09 0.85 0.18 1.30 0.46 1.58 1.28 1.93 0.03 0.40 0.02 0.38 0.02 0.45 0.01 0.75 2.20 19.44 ''Based on Marschner and Richter (1973). ^Each seedling was supplied with 1 meq T^ of labeled nutrient solution to the root zone 12-15 cm from the root tip. The remainder of the root system was supplied with the same solution in which the nutrients were not labelled. counterflow of water in the xylem may also occur, for example, from low-transpiring fruits back to the leaves (Lang and Thorpe, 1989; Section 3.4). In contrast to the xylem, long-distance transport in the phloem takes place in the living sieve tube cells and is bidirectional. The direction of transport is determined primarily by the nutritional requirements of the various plant organs or tissues and occurs, therefore, from source to sink (Chapter 5). In addition, phloem transport is an important component in cycling of mineral nutrients between shoots and roots (Section 3.4) and for signal conductance of the nutritional status of the shoots (Section 2.5.6). Mineral elements can also enter the phloem in the roots and thus be translocated bidirectionally. The translocation of different mineral elements taken up by a particular zone of the root varies markedly during long-distance transport from the zone of supply, as shown in Table 3.1 for maize seedlings. For the reasons already mentioned, long-distance transport from the zone of supply to the root tip must take place in the phloem. Whereas "̂ Ĉa is rapidly translocated to the shoot, the translocation of ^̂ Na toward the shoot is severely restricted. The steep gradient in the ^̂ Na content of the root sections in the direction of the shoot (basipetal) reflects resorption by the surrounding root tissue and is a typical feature of so-called natrophobic plant species (Section 10.2). Some ^̂ Na has also been translocated via the phloem to the root tip. In contrast, "̂ K̂ is quite mobile both in the xylem and in the phloem, and a markedly high proportion of the potassium taken up in more basal root zones is translocated via the phloem toward the root tip, which acts as a sink for this mineral nutrient. Long-Distance Transport in the Xylem and Phloem and Its Regulation 81 Table 3.2 Xylem Volume Flow (Pressurized Exudation at 100 kPa) and Mineral Element Concentrations in the Xylem Sap of Soil-Grown Nodulated Soybean During Reproductive Stage" Parameter Sap volume (ml(50min)-^ per plant) Mineral element concentration K(mM) Mg (mM) Ca (mM) P(mM) S(mM) B (mM) Zn (JUM) Cuijuu) Full pod extension 1.43 6.1 3.8 4.8 2.5 1.8 1.0 23.0 2.7 Plant development stages Early-mid podfill 1.13 5.0 2.6 3.9 1.6 1.6 1.5 29.0 3.6 Late podfill 0.94 4.0 1.9 3.9 0.9 2.1 1.6 32.0 2.8 Early leaf yellowing 0.43 2.4 1.2 2.2 0.4 1.5 3.2 42.0 6.9 ''Based on Nooden and Mauk (1987). During long-distance transport, mineral elements and organic solutes are transferred between the xylem and phloem by extensive exchange processes, referred to as loading and unloading. The transfer is mediated by specific cells called transfer cells (Pate and Gunning, 1972). Despite this interchange, and internal cycling, mineral nutrients, such as phosphorus, suppKed to only one part of the root system (lateral or seminal roots) are transported preferentially to those parts of the shoots that have direct vascular connections with particular root zones (Stryker etal., 1974). This distribution pattern is especially important for the mineral nutrition of trees that are suppUed with fertilizer in a localized area of the root system. 3.2 Xylem Transport 3.2.1 Composition of the Xylem Sap The composition and concentration of mineral elements and organic solutes in the xylem sap depend on factors such as plant species, mineral element supply to the roots, assimilation of mineral nutrients in the roots and nutrient recycling. Composition and particularly concentration of solutes are also strongly influenced by the degree of dilution by water (Section 2.9) and are therefore dependent on the transpiration rate and the time of day. Composition and concentration of xylem sap also change typically during the ontogenesis of plants (T^ble 3.2). In soybean during the reproductive stage 84 Mineral Nutrition of Higher Plants Bean Sugar beet Fig. 3.2 Distribution of sodium in bean {Phaseolus vulgaris L.) and sugar beet {Beta vulgaris L.) 24 h after 5 mM ^^NaCl was supplied to the roots. Autoradiogram. lower stem, whereas in natrophilic species (e.g. sugar beet) translocation into the leaves readily occurs (Fig. 3.2). This restricted upward Na^ translocation is caused by selective Na^ accumulation in the xylem parenchyma cells (Rains, 1969; Drew and Lauchli, 1987) together with re translocation into the roots (Fig. 3.10). In castor bean these two components led to a depletion in the Na^ concentration from 0.8 to 0.2 mM in the upward-moving xylem stream (Jeschke and Pate, 1991b). Resorption of Na^ from the xylem sap is therefore an effective mechanism of restricting translocation to the leaf blades. This mechanism, however, is not necessarily advantageous for the salt tolerance of plants (Drew and Lauchli, 1987; Jeschke and Pate, 1991b; see also Section 16.6) and is also a disadvantage in forage plants. For animal nutrition the sodium content of the forage should be at least 0.2%. As shown in Table 3.3, in Lolium perenne and Trifolium repens, Na^ is readily translocated to the Table 3.3 Effect of Sodium Fertilizer on the Sodium Content of Roots and Shoots of Pasture Plants" Plant species Lolium perenne Phleum pratense Trifolium repens Trifolium hybridum Na Content (% dry wt) Without Na fertilizer Roots 0.03 0.10 0.27 0.45 Shoots 0.26 0.04 0.22 0.03 With Na fertilizer Roots 0.06 0.28 0.77 0.77 Shoots 1.16 0.38 1.96 0.22 "Based on Saalbach and Aigner (1970). Long-Distance Transport in the Xylem and Phloem and Its Regulation 85 Table 3.4 Distribution of Molybdenum in Bean and Tomato Plants Supplied with Molybdenum in the Nutrient Solution^ Molybdenum content (/^gg-^drywt) Plant parts Bean Tomato Leaves Stems Roots 85 210 1030 325 123 470 "Concentration of molybdenum in solution: 4 mg 1 ^ Based on Hecht-Buchholz (1973). shoots, whereas in Phleumpratense and Trifolium hybridum this translocation is rather restricted. It is evident that in order to increase the sodium content of forage selection of suitable plant species is more important than the application of sodium fertilizers. Resorption from the xylem sap in roots and stems can also be a determining factor in the distribution of micronutrients in plants. In certain species, such as bean and sunflower, molybdenum is preferentially accumulated in the xylem parenchyma of the roots and stems. In these species a steep gradient occurs in the molybdenum concen- trations from the roots to the leaves (Table 3.4). In contrast, in other species, such as tomato, molybdenum is readily translocated from the root to the leaves. In accordance with this finding, when the molybdenum supply in the nutrient medium is high, toxicity occurs much earlier in tomato than in bean or sunflower (Hecht-Buchholz, 1973). 3.2.2,2 Release or Secretion The composition of the xylem sap along the transport pathway can also be changed by the release or secretion of solutes from the surrounding cells. For example, in nonlegumes supphed with nitrate, the nitrate concentration in the xylem sap decreases as the path length increases, whereas the concentration of organic nitrogen, glutamine in particular, increases (Pate et al., 1964). In nodulated legumes (where N2 fixation occurs), on the other hand, the ratio of amides to amino acids is shifted in favor of the amino acids (Pate etal, 1979). Besides these specific aspects of nitrogen translocation, the release or secretion of mineral nutrients from the xylem parenchyma (and stem tissue in general) is of major importance for the maintenance of a continuous nutrient supply to the growing parts of the shoots. In periods of ample supply to the roots, mineral nutrients are resorbed from the xylem sap, whereas in periods of insufficient root supply they are released into the xylem sap. Changes in the potassium and nitrate contents of the stem base reflect this functioning of the tissues along the xylem in response to changes in the nutritional status of a plant. From this information a rapid test for nitrate in the stem base has been developed as a means for recommending rates of nitrogen fertilizer application (Section 12.3.8). 86 Mineral Nutrition of Higher Plants 3.2.23 Xylene Unloading in Leaves Despite resorption along the pathway in the stem most of the solutes and water are transported in the xylem vessels into the leaves. Here water is preferentially trans- ported in the major veins to sites of rapid evaporation such as leaf margins, or from the vein endings mainly via symplasmic movement towards the stomata (Canny, 1990). Although the bundle sheath walls of the veins are suberized in grass leaves of C3 and C4 species, they do not provide a barrier against apoplasmic flux of water and solutes (Eastman et al., 1988). Depending on the concentration and composition of solutes in the xylem sap entering the leaf, and the rate of water loss by transpiration, along its stream through the leaf, the solute concentration may be enriched manyfold at predictable sites, as for example, the leaf edges. This is particularly true when mineral element concentrations are high in the root medium (e.g. saHne substrates) and for mineral elements such as boron and siHcon (Section 3.2.4). Unless some of this excessive solute accumulation at the terminal sites of the transpiration stream is not removed, for example, by loading into the phloem, by guttation, as has been shown for boron (Oertli, 1962) or in epidermal glands in halophytes (Fitzgerald and AUaway, 1991), necrosis on the tips or margins of leaves occur (Fig. 3.7). This is a reflection of insufficient resorption of solutes along the pathway of the transpiration stream in the leaf. Prevention of excessive solute accumulation in the leaf apoplasm by mechanisms other than uptake by the leaf cells can be achieved by the formation of salts of low solubility in the apoplasm. This strategy seems to be used particularly for the removal of soluble calcium in gymnosperms (Fink, 1991a). Calcium oxalate crystals are abundant in the needles of various gymnosperms in the cell walls of the mesophyll and particularly of the phloem and in the outer wall of the epidermis (Fig. 3.3). This mechanism of precipitation seems to be a safe way of coping with a continuous xylem import of calcium which is scarcely exported in the phloem (Section 3.4.3) and where the ionic concentrations in the symplasm have to be kept very low. The origin of oxalic acid in the apoplasm is unknown. Oxalic acid may be released from the cytoplasm or be formed in Fig. 3.3 Calcium oxalate crystals in the apoplasm of needles. (Left) Micrograph from the phloem of a needle from Juniperus chinensis; (right) micrograph of a stomatal pore of a needle from Picea abies (L.) Karst. (Courtesy of S. Fink, 1991a,c.) Long-Distance Transport in the Xylem and Phloem and Its Regulation 89 1. Plant age. In seedlings and young plants with a low leaf surface area, enhancement effects of transpiration are usually absent; water uptake and solute transport in the xylem to the shoots are determined mainly by the root pressure. As the age and size of the plants increase, the relative importance of the transpiration rate, particularly for the translocation of mineral elements increases. 2. Time of day. In leaves up to 90% of the total transpiration is stomatal. During the hght period, transpiration rates and thus the potential enhancement of uptake and translocation of mineral elements are much higher than during the dark period. Short- term transient falls in the translocation rates of mineral elements at the onset of the dark period reflect the change from transpiration-mediated to root pressure-mediated xylem volume flow (Crossett, 1968). A consistent and synchronous diurnal pattern in transpiration rate and uptake rate of potassium and nitrate (Le Bot and Kirkby, 1992) is probably caused by changes in carbohydrate availability in the roots or feedback control of uptake. Nodulated legumes show a particular diurnal pattern in shoot transport of fixed nitrogen. The sharp decrease in transpiration-driven xylem volume flow during the dark period is compensated for by a sharp increase in the concentration of fixed nitrogen (as ureides, see Chapter 7) in the xylem sap, thus keeping the total xylem transport rate of fixed nitrogen constant throughout the Hght/dark cycle (Rainbird etal., 1983). 3. External concentration. It is well known that an increase in the concentration of mineral elements in the nutrient medium may enhance the effect of transpiration rate on the uptake and translocation of mineral elements. This is most Ukely the result of transport as shown in schemes A and C in Fig. 3.5. Usually, translocation rates are more responsive to different transpiration rates than are uptake rates, as shown for sodium in Table 3.5. The effect of transpiration on potassium is negUgible in comparison with that Table 3.5 Effect of Transpiration Rate of Sugar Beet Plants on Uptake and Translocation of Potassium and Sodium from Nutrient Solutions'"'^ External concentration (mM) 1K++ INa-' 10 K-" + 10 Na"" 1 K"" + 1 Na-' 10 K-" + 10 Na-' Potassium Sodium Low High Low High transpiration transpiration transpiration transpiration Uptake rate (^mol per plant (4 h)~^) 4.6 4.9 8.4 11.2 10.3 11.0 12.0 19.1 Translocation rate (//mol per plant (4 h)~^) 2.9 3.0 2.0 3.9 6.5 7.0 3.4 8.1 "Based on Marschner and Schafarczyk (1967) and W. Schafarczyk (unpublished). ^Transpiration in relative values: low transpiration = 100; high transpiration = 650. 90 Mineral Nutrition of Higher Plants on sodium. This difference corresponds to the differences in the uptake isotherms of these elements at increasing external concentrations (Fig. 2.22). At low external concentrations the nitrate flux in the xylem of maize plants is also not affected by decreasing the transpiration rate to 50%, and a reduction in transpiration rate to 20% being required before a major decline in nitrate flux becomes apparent (Shaner and Boyer, 1976). 4. Type of mineral element. Under otherwise comparable conditions (e.g. plant age and external concentration), the effect of transpiration rates on the uptake and transport follows a typically defined ranking order of mineral elements. It is usually absent or only low for potassium, nitrate and phosphate but may become significant for sodium (Table 3.5) or calcium. As a rule, transpiration enhances the uptake and translocation of uncharged molecules to a greater extent than that of ions. There is a close relationship between transpiration rate and uptake rates of certain herbicides (Shone et al., 1973). The uptake and translocation of mineral elements in the form of molecules is of great importance in the cases of boron (boric acid; Fig. 2.13) and silicon (monosilisic acid; Jones and Handreck, 1965; but see Section 10.3). A close correlation between transpiration and the uptake of silicon is shown for oat plants in Table 3.6. There is perfect agreement between silicon content measured in plants and that predicted from the transpiration values (water loss times silicon concentration in the soil solution). Silicon accumulation in the shoot dry matter may therefore be a suitable parameter for calculations of the water use efficiency (WUE; kg dry matter produced/ kg water transpired) in field-grown cereals grown under rainfed conditions (Walker and Lance, 1991). However, this parameter is unsuitable, for example, in plants grown at different irrigation regimes (Mayland et al., 1991) as well as in plants grown in nutrient solution (Jarvis, 1987), or when different genotypes within a species such as barley are compared (Nable etal., 1990b). Even in plants where close correlations between transpiration and silicon uptake are found, roots are not freely permeable to the radial transport of siUcon. In wheat in endodermal cells large silicon depositions are found increasing from apical to basal root zones (Hodson and Sangster, 1989c) and such large deposition in the endodermis is typical for field-grown cereals (Bennett, 1982). Table 3.6 Measured and Calculated Silicon Uptake in Relation to Transpiration (Water Consumption) of Oat Plants'* Harvest Transpiration Measured uptake Calculated Si uptake^ after days (ml per plant) (mg per plant) (mg per plant) 44 67 3.4 3.6 58 175 9.4 9.4 82 910 50.0 49.1 109 2785 156.0 150.0 "From Jones and Handreck (1965). ^Silicon concentration in the soil solution: 54 mg T^ Long-Distance Transport in the Xylem and Phloem and Its Regulation 91 The absence of effects of reduced transpiration rates on the root to shoot transport of mineral nutrients may indicate a high proportion of xylem to phloem transfer in the stem tissue, or a corresponding increase in xylem sap concentrations of the mineral nutrients. Alternatively, the involvement of a nontranspirational component of xylem transport, namely the so-called Munch-counterflow has been stressed recently (Tanner and Beevers, 1990). This component is based on recycling of water derived from solute volume flow in the phloem from shoots to roots (Section 3.4.4) and the release of this water, and some of the solutes (recycled fraction) into the xylem. According to Tanner and Beevers (1990) the calculated amounts of water recycled in this manner vary between 9% (at high transpiration rates) and 30% (at low transpiration rates) of the total water uptake by the roots of maize plants. Values in this high order have been questioned for various reasons by Smith (1991). Nevertheless, recycUng of water in plants must be taken into account in relation to recycling of mineral nutrients in general and distribution of calcium within the shoot in particular (Section 3.4.3). 3.2.4 Effect of Transpiration Rate on Distribution within the Shoot The long-distance transport of a mineral element exclusively in the xylem should be expected to give a distinct distribution pattern in the shoot organs that depends on both transpiration rates (e.g., ml g~̂ dry weight each day) and duration of transpiration (e.g. age of the organ). This is true, for example, for manganese (McCain and Markley, 1989) where at the same plant (maple tree) and similar leaf age the *sun leaves' (high transpiration rates) have much higher manganese contents in their dry matter than 'shade leaves' (low transpiration rates). Both the distribution and content of silicon usually reflect the loss of water from the various organs. The siUcon content increases with leaf age and is particularly high in spikelets of cereals such as barley. Even within a certain tissue, silicon distribution resembles the pathway of transpiration flow in the apoplasm. Sihcon is deposited in the walls of the epidermis cells (Hodson and Sangster, 1988) or in the pericarp and outer aleurone layer of grass seeds such as Setaria italica (Hodson and Parry, 1982). The distribution of boron is also related to the loss of water from the shoot organ, as shown by the boron distribution in shoots of rape in response to an increasing boron supply (Fig. 3.6). The typical gradient in the transpiration rates in the shoot organs (leaves > pods » seeds) corresponds to the gradient in boron content. Even for a particular leaf, an excessive supply of boron creates a steep gradient in the boron content: petioles < middle of the leaf blade < leaf tip (Oertli and Roth, 1969). Necrosis on the margins or leaf tips is therefore a typical symptom of boron toxicity (Fig. 3.7). In salt-affected plants the visible symptoms of toxicity (e.g., by chloride) are often quite similar, reflecting the transpiration-mediated distribution pattern within the shoot and its organs. Frequently, a close positive correlation is observed between calcium distribution and the transpiration rates of shoot organs. This is shown, for example, by the low calcium content in the dry matter of low transpiring fleshy fruits (<0.3% calcium) as compared with that of the leaves (3-5% calcium) in the same plant. A lowering of the transpiration rate further decreases the calcium content of fruits (Table 3.7). The effect of transpi- ration on magnesium is much lower than its effect on calcium, and that on potassium is