Mineral Nutrition and Plant Disase

1 The Chemistry of Plant Nutrients in Soil Samira H. Daroub and George H. Snyder University of Florida, Institute of Food and Agricultural Sciences Everglades Research and Education Center, Belle Glade

Introduction

Oxygen, on the other hand, can be absorbed by both leaves and roots, but always in the gaseous diatomic form, O2. Oxygen in soil is generally present at much lower concentrations than in the atmosphere, because of consumption by soil microorganisms. For this reason, plant root activity can be limited by the availability of O2. Oxygen is especially limited when the pore spaces between soil particles are filled with water, since O2 diffusion in water is only 1 × 10−4 times that of diffusion in air. Under low-O2 conditions, terrestrial plant roots may have difficulty absorbing nutrients and water.

Aquatic plants, on the other hand, contain parenchyma tissue that transmits O2 from the leaves to the roots, so the roots can function even in flooded soils.

Hydrogen enters plants as a component of water (H2O). As long as sufficient water is present to maintain plant turgor, sufficient H is available for metabolic needs.

At least 16 elements are considered to be essential for plant growth and reproduction, and a few others are beneficial for certain plants. Those taken up and utilized by plants in the greatest quantities are termed macronutrients. Micronutrients, while equally essential, are utilized by plants in much smaller quantities. Plants obtain three of the macronutrients, carbon (C), hydrogen (H), and oxygen (O), from air and water. The others are absorbed from the soil in ionic forms, some as cations and others as anions, which interact with the soil, often in complex ways. The ability of some nutrients to be absorbed by plants (i.e., plant availability) is largely dominated by the activities of soil microorganisms, whereas the plant availability of other nutrients is governed more by inorganic chemical reactions. The purpose of this chapter is to elucidate the processes in soil that affect the plant availability of essential elements.

Nitrogen

The Macronutrients

Most nitrogen (N) in soils is present in the organic form as a component of humus. As such, N is not available for plant absorption (uptake). Plant roots absorb nitrogen in its ionic forms: ammonium (NH4+), nitrite (NO2−), and nitrate (NO3−). The NO2− form is quite transitory in soils and is toxic to plants, so there is very little uptake. Ammonium results from the decomposition of humus and other forms of soil organic matter by soil microorganisms. The rate of decomposition is regulated by factors that affect microbial activity,

Carbon, Oxygen, and Hydrogen In the early 1800s, respected scientists, such as A. von Thaer in Germany, Sir Humphry Davy in England, and J. J. Berzelius in Sweden, considered that plants absorbed C through their roots largely from soil organic matter, i.e., humus (Russell 1961). Today it is known that plants obtain C through their leaves from atmospheric carbon dioxide.

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such as soil temperature, moisture, O2, and pH. For this reason, the supply of N for plant uptake can be limited when soil temperature, moisture, O2, and pH are low or when temperatures and moisture are too high for optimal microbial activity. In well-aerated soils, NH4+ is rapidly converted to NO2− and then NO3−, so most N absorbed by plants is in the NO3− form. Considerable soil acidity is created in the conversion of NH4+ to NO3−. Because NO3− is rapidly absorbed by plants and is also readily leached, soils generally contain little NO3− in the root zone. Continual replenishment of N is required to maintain rapid plant growth. When O2 is limiting, as in very wet soils, microorganisms convert NO3− to gaseous forms, such as nitric oxide (NO), nitrous oxide (N2O), and dinitrogen (N2), resulting in N losses from the soil. Nitrogen can be added to soils as fertilizer in organic, NH4−, and NO3− forms. In addition, certain microorganisms are capable of converting atmospheric N (N2), which is not available to plants, to organic forms that are available to plants. The process is termed N2 fixation. Some N2 fixation is accomplished by free-living microorganisms, but most biological N2 fixation in soils occurs as the result of symbiotic interaction of bacteria and legumes.

Phosphorus Phosphorus (P) is present in both organic and inorganic forms in soils. While the percentages of each can vary widely, substantial amounts of both forms generally are present. Phosphorus is absorbed by plant roots mainly as phosphate anions (H2PO4− and HPO42−). The concentration of P in the soil solution is very low, generally ranging from 0.001 mg L−1 in very infertile soils to 1 mg L−1 in heavily fertilized soils

Figure 1.1. Effect of soil pH on the relative abundance of various chemical forms of phosphorus. Phosphates available to plants constitute relatively little of the total phosphorus in the soil and are most abundant at pH 6–7. (Reprinted from Brady and Weil 2002, © 2002, by permission of Pearson Education, Inc., Upper Saddle River, N.J.)

(Brady and Weil 2002). For uptake to occur, P in soil organic matter must be converted to orthophosphate anions by soil microorganisms (a process known as mineralization). The release of P from organic matter, like that of N, depends on soil temperature, moisture, O2, and pH. The amount of plant-available P in soils is generally low, because phosphate anions are quite chemically active. In acid soils in which cations of metals such as aluminum (Al), iron (Fe), and manganese (Mn) are abundant, insoluble complexes are formed with P. In alkaline soils, P forms relatively insoluble compounds with calcium (Ca). Phosphate ions are strongly adsorbed by silicates in soils of near neutral pH. Consequently, while soils may contain considerable quantities of P, most of it is present in forms that are not found in the soil solution and available for plant uptake (Figure 1.1). Fertilizer P is generally in the form of highly soluble monocalcium phosphate, Ca(H2PO4)2. Upon addition to soil, substantial amounts of the highly reactive H2PO4− form insoluble compounds with Al, Fe, or Ca, depending on soil pH (Figure 1.1). Some plant-available P is eventually released by the slow dissolution of these compounds.

Potassium Unlike N and P, almost all potassium (K) in soils is present in inorganic forms. Sandy soils, being composed primarily of quartz (SiO2), frequently contain little K, but many soils contain very large amounts of it.

However, most of the K in these soils is present as relatively insoluble minerals, such as feldspars and micas, or is “fixed” in the mineral lattice of certain clays, such as hydrous micas (also known as illite). Consequently, K deficiency can be observed in plants growing in soils containing considerable amounts of total K. Potassium is absorbed by plants in the cationic form (K+). Soil humus and soil clays generally are negatively charged, so K+ is adsorbed by these materials. Such K is termed exchangeable, because plants exchange secreted hydronium ions (H3O+) for adsorbed K+. They also take up K+ that is dissolved in the soil solution.

Once present in the soil solution as K+, K forms few insoluble compounds. However, hydrous mica clays can reduce soil solution K by entrapping it in their mineral lattice. Potassium in fertilizers is generally in the form of soluble K salts, such as potassium chloride, potassium sulfate, or potassium magnesium sulfate.

Calcium and Magnesium Both calcium (Ca) and magnesium (Mg) are absorbed by plants in the divalent cationic form (Ca2+ and Mg2+).

Like K, most Ca and Mg exist in soil in mineral forms.

THE CHEMISTRY OF PLANT NUTRIENTS IN SOIL

However, the predominant forms in alkaline soils are the carbonates, CaCO3 and MgCO3. In comparison to N and P, relatively little plant-available Ca and Mg originates from soil humus.

In addition to carbonates, minerals such as hornblende, augite, anorthite, olivine, biotite mica, and gypsum supply soil Ca and Mg as they weather.

Highly acid soils may be quite low in plant-available Ca and Mg. Clays, being negatively charged, adsorb Ca2+ and Mg2+, often preferentially to other cations.

Therefore, a substantial quantity of the Ca and Mg in soils is in the readily available exchangeable form. As was stated above, Ca reacts with P to form relatively insoluble compounds, but the reaction generally has more consequence for limiting P availability than for Ca. Calcium and sometimes Mg fertilization is often accomplished by adding lime or dolomite to soils to increase the soil pH. When an increase in pH is not desired, Ca and Mg may be supplied in the form of sulfates.

Sulfur Like N and P, sulfur (S) is present in soils in both organic and inorganic forms. In temperate humid regions, over 90% of the S in soils is present in soil organic matter. However, S is absorbed by plants as the sulfate anion, SO42−. Consequently, organically bound S must be released by microbial decomposition to be available for plant uptake, so the factors that affect microbial activity (temperature, moisture, O2, and pH) affect S availability. In soils with very poor aeration, S is present in reduced forms, such as S0, or as sulfides (S2−). The rotten-egg smell of hydrogen sulfide is a classic indicator of very poor soil aeration. Mineral sulfides include the well-known compound fool’s gold (iron pyrite). In well-aerated soil, elemental S readily oxidizes to SO42−, which increases the acidity of the soil. Most bacteria function poorly under highly acidic conditions, but those that oxidize sulfur continue to function in spite of the acidity that results from the production of SO42−.

Sulfates can be adsorbed somewhat on clay surfaces, especially in acid soils high in Fe and Al oxides, and by the clay mineral kaolinite, but adsorbed sulfates represents a small component of the total sulfur in most agricultural soils. Other than forming gypsum with Ca in arid soils, S undergoes few inorganic reactions that limit its availability to plants.

In industrial areas, considerable S may be added to soils by way of airborne contaminants, such as those produced during the combustion of fossil fuels.

When S fertilization is required, elemental S or various SO42− salts, such as gypsum or magnesium sulfate, may be used.

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The Micronutrients Micronutrients are elements essential for plant growth, although required only in small quantities. These elements are iron, zinc, copper, manganese, boron, molybdenum, and chlorine. Cobalt and nickel have been established as essential elements for some but not all higher plants. Cobalt is essential for N2 fixation by legumes. Nickel is essential for urease, hydrogenases, and methyl reductase and for urea and ureide metabolism, to avoid toxic levels of these nitrogen fixation products in legumes (Brady and Weil 2002). Other elements that may be beneficial include sodium, silicon, selenium, and vanadium.

Iron Iron (Fe) is the fourth most abundant element in the lithosphere. It is geochemically unique in its ability to form numerous stable compounds both with S and with O plus Si. Most of the Fe in soil occurs in primary minerals, clays, oxides, and hydroxides. The primary minerals in which Fe are present include ferromagnesian silicates, such as olivine, augite, hornblende, and biotite (Mengel and Kirkby 2001). Weathering of these minerals produces secondary FeIII minerals, such as hematite (α-Fe2O3) and geothite (α-FeOOH). The solubility of the FeIII oxides is extremely low at high pH. Iron deficiency is most often observed in high-pH and calcareous soils of arid regions, but it also occurs in acidic soils that are very low in total Fe. The reduction-oxidation (redox) potential of soils also affects Fe solubility. Iron compounds become more soluble as FeIII oxides are reduced to Fe2+ (dissolved in the soil solution) when the oxygen supply in soils is low, as in wet soils containing decomposable organic matter.

The concentration of soluble Fe in soils is extremely low in comparison with the total Fe concentration.

Iron in the soil solution is found as Fe2+, Fe (OH)2+, Fe(OH)2+, and Fe3+, with Fe3+ being dominant. Iron solubility is largely controlled by the solubility of hydrous FeIII oxides. These give rise to Fe3+ and its hydrolysis species (Lindsay 1979): Fe3+ + 3OH − ↔ Fe(OH)3 (solid) For every unit increase in pH, the Fe3+ concentration decreases 1,000-fold. Over the normal pH range in soils, soil solution Fe is not sufficient to meet plant requirements, even in acidic soils. Numerous natural organic compounds in soil are able to complex or chelate Fe3+ and other micronutrients and therefore increase the availability of these micronutrients in the soil solution. Chelates are soluble organic compounds that bond with metals such as Fe, Zn, Cu, and Mn,

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CHAPTER 1

increasing the solubility of these metals and their supply to plant roots. The Fe chelates (siderophores) are highly soluble and are stable over a wide range of pH. Natural organic chelates in soils are products of microbial activity and degradation of soil organic matter and plant residues. Most animal wastes contain small

Table 1.1. Chemical formulas of common synthetic and natural chelates.a Formula Ethylenediaminetetraacetic acid Diethylenetriaminepentaacetic acid Ethylenediaminedi-o-hydroxyphenylacetic acid Citric acid Oxalic acid Pyrophosphoric acid a

Abbreviation

C10H16O8N2 EDTA C14H23O10N3 DTPA C18H20O6N2 C6H8O7 C2H2O4 H4P2O7

EDDHA CIT OX P2O7

Source: Norvell 1972. Used by permission.

Figure 1.2. Stability of Fe chelates in the soil solution.

Equilibrium between H+, Ca2+, Mg2+, Al3+, and Fe3+ is assumed, and the concentration of each chelating agent is assumed to be 10−4 M. CDTA = cyclohexanediaminetetraacetic acid.

CIT = citric acid. DTPA = diethylenetriaminepentaacetic acid. EDDHA = ethylenediaminedi-o-hydroxyphenylacetic acid. EDTA = ethylenediaminetetraacetic acid. EGTA = ethyleneglycol-bis(2-aminoethylether)-tetraacetic acid.

HEDTA = hydroxyethylethylenediaminetriacetic acid.

NTA = nitrilotriacetic acid. OX = oxalic acid. (Reprinted from Norvell 1972, by permission)

quantities of plant-available Fe. Synthetic chelates of Fe, Zn, Cu, and Mn are often applied to soils as fertilizers. Common natural and synthetic chelates are listed in Table 1.1. Chelation increases the solubility and transport of micronutrients. The stability of chelates is greatly affected by pH (Norvell 1972) (Figure 1.2).

Fe-EDDHA (ethylenediaminedi-o-hydroxyphenylacetic acid) is stable over the pH range of 4 to 9, while chelates like Fe-EDTA (ethylenediaminetetraacetic acid) and Fe-DTPA (diethylenetriaminepentaacetic acid) are not stable at high pH. Abundant Ca in highpH soils will easily replace the Fe on EDTA or DTPA, and the Fe will precipitate as an oxide.

Manganese Manganese is found in various primary rocks, particularly ferromagnesian (Fe-Mg) materials. The weathering of primary rocks releases Mn, which forms secondary minerals, including pyrolusite (MnIVO2) and manganite (MnIIIO(OH)) (Mengel and Kirkby 2001).

Manganese in the soil exists as Mn2+ dissolved in the soil solution, exchangeable Mn2+, organically bound Mn, and the Mn oxides. Factors affecting the solubility of Mn include pH, redox, and complexation.

Mn solubility decreases at high pH, as a result of its precipitation as MnO2. According to the prevailing redox potential Mn may be present in di-tri and tetravalent form. Waterlogging will reduce O2 and lower oxidation potential, which increases soluble Mn2+, especially in acidic soils. In extremely acidic soils, Mn2+ solubility can be high enough to cause toxicity in sensitive plant species. Under anaerobic conditions Mn2+ is produced mainly by microbial respiration functioning as an electron (e−) donor for MnIII and MnIV compounds, and thus Mn oxides are dissolved (Mengel and Kirkby 2001).

Most organisms that reduce Fe3+ also reduce Mn4+ (Paul and Clark 1996). The availability of Mn2+ can be strongly influenced by reactions with organic matter.

The low availability of Mn in soils with high organic matter content is attributed to the formation of unavailable chelated Mn2+ compounds. Manganese can be held in unavailable organic complexes in peats or muck soils.

Sustained adequate Mn nutrition of Bermudagrass turf was achieved at high pH with Mn applied to plots, in which fungal activity was suppressed by repeated fungicidal drenches (Snyder et al. 1979). Numerous fungal species have been implicated in the oxidation of Mn2+ to less soluble forms in the pH range of 7 to 8.

Zinc Zinc (Zn) is found in igneous and sedimentary rocks and in the lattice structure of primary and secondary minerals. Soils originating from basic igneous

THE CHEMISTRY OF PLANT NUTRIENTS IN SOIL

rocks are high in Zn. In contrast, soils derived from siliceous parent materials are particularly low in Zn. On a global scale, more than 30% of agricultural soils are deficient in Zn. Common Zn-containing minerals are franklinite (ZnFe2O4), smithsonite (ZnCO3), and willemite (ZnSiO4) (Havlin et al. 2005). Zinc is dissolved in the soil solution in ionic or complex forms and may be found on exchange sites of clay minerals and organic matter or adsorbed on solid surfaces as Zn2+, ZnOH+, or ZnCl+.

The zinc concentration in the soil solution is very low, ranging between 2 and 70 ppb. Zinc mineral solubility in soils is represented by “soil Zn,” which could be a mixture of different compounds. More than half of the Zn2+ in solution is complexed by organic matter.

Above pH 7.7, ZnOH+ becomes the most abundant species in solution (Havlin et al. 2005). Zinc solubility is highly pH-dependent and is very low at high soil pH. It is particularly low when CaCO3 is present, because of specific adsorption of Zn2+ to and occlusion by carbonates (Mengel and Kirkby 2001). Calcareous soils often have poor Zn availability, as a result of Zn occlusion by carbonates.

Copper Copper (Cu) is found in igneous and sedimentary rocks. Malachite (Cu(OH)2CO3) and cupric ferrite (CuFe2O4) are the important Cu-containing primary minerals (Havlin et al. 2005). Secondary Cu minerals include oxides, carbonates, silicates, sulfates, and chlorides, but most of them are too soluble to persist. “Soil Cu” represents the solubility of Cu in most soils and is very close to the solubility of CuFe2O4. A significant fraction of soil Cu is occluded or buried in clay minerals as Fe, Al, and Mn oxides. Copper is present as an impurity in CaCO3 and MgCO3 in arid soils and in Al(OH)3 and Fe(OH)3 in acidic soils.

Besides the Cu present in minerals, Cu is dissolved in the soil solution, adsorbed to soil, and associated with organic matter. The Cu concen-tration in the soil solution is usually very low, ranging between 10−8 and 10−6 M. The dominant solution species are Cu2+ at pH < 7 and Cu(OH)20 at pH > 7 (Havlin et al. 2005).

The concentration of Cu2+ in the soil solution decreases sharply with increasing pH. Copper is specifically adsorbed by carbonates, layer silicates, clays, organic matter, and Fe, Mn, and Al hydrous oxides (Mengel and Kirkby 2001). With the exception of Pb2+ and Hg2+, Cu2+ is the most strongly adsorbed of all of the divalent metals on Fe and Al oxides (Havlin et al. 2005).

Cu adsorption increases with increasing pH. Most of the soluble Cu in surface soils is organically complexed and is more strongly bound to organic matter than any other micronutrient. Copper added

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to the soil in copper-containing fertilizers is thus largely restricted to the upper soil horizons and can reach high or even toxic levels. Copper deficiency occurs primarily on humus-rich soils, which strongly bind Cu2+.

Boron The total boron (B) concentration in soils is in the range of 20–200 mg kg−1, most of which is inaccessible to plants. The available, hot-water-soluble fraction in soils adequately supplied with B ranges from 0.5 to 2.0 mg L−1 (Mengel and Kirkby 2001). Boron exists in four major forms in the soil: (1) in rocks and minerals, (2) as boric acid (H3BO30) and B(OH)4− in the soil solution, (3) adsorbed on clay surfaces and on Fe and Al oxides, and (4) combined with organic matter (Havlin et al. 2005). Boron is the only nonmetal among the micronutrients. The main B-containing mineral in the soil is tourmaline, a borosilicate. Tourmaline releases little B, as it is resistant to weathering. Undissociated H3BO30 is the predominant species expected in the soil solution at pH 5 to 9. Boric acid is undissociated in the soil solution, and thus B can easily be leached from the soil. In soils of arid and semiarid regions, B may accumulate to toxic concentrations in the upper soil layer, because of lack of leaching due to low rainfall.

The availability of B is affected by pH, organic matter, and soil texture. Its availability decreases as the soil pH increases and is dramatically reduced above pH 6.3. Organic matter is a large potential source of plant-available B in soils. Coarse-textured, welldrained, sandy soils are low in B. Sodium tetraborate (Na2B4O7 · 5H2O) is the most commonly used source of B for fertilization. Boron fertilizers should be applied uniformly to soil, because of the narrow range between deficiency and toxicity (Havlin et al. 2005).

Molybdenum The main forms of molybdenum (Mo) in soil are Mo in primary and secondary minerals; exchangeable Mo held by Fe and Al oxides; Mo in the soil solution, predominantly as the molybdate ion (MoO42−); and organically bound Mo (Havlin et al. 2005). The minerals in the soil controlling the MoO42− concentration are PbMoO4 and CaMoO4. The Ca mineral predominates in both acidic soil and calcareous soil. In the soil solution, Mo occurs predominantly as MoO42− at pH > 4. The Mo concentration in the soil solution depends on the soil pH and the total Mo content of the soil. As the pH falls, the Mo concentration in the soil solution decreases.

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Mo in the soil solution is in an anionic form, unlike other heavy metal nutrients, and molybdate resembles phosphate and sulfate in its behavior in soils (Mengel and Kirkby 2001). Molybdate is adsorbed by sesquioxides and clay minerals in a similar manner to phosphate. Adsorption of Mo in soils, like that of phosphate and sulfate, is strongly pH-dependent, increasing with decreasing pH. This reduces the availability of Mo at low pH. Molybdenum availability, unlike that of other micronutrients, increases about 10-fold per unit increase of pH.

Chlorine In nature, the chloride ion (Cl−) is widely distributed and subject to rapid recycling. Nearly all the Cl− in soils exists in the soil solution and is not adsorbed by minerals. Appreciable Cl− leaching can occur in well-drained soils. Most of the soil Cl− commonly exists as soluble salts, such as NaCl, CaCl2, and MgCl2. The quantity of Cl− in the soil solution may range from 0.5 mg kg−1 or less to more than 6,000 mg kg−1 (Havlin et al. 2005).

Most Cl− in soils originates from salts trapped in parent material, from marine aerosols, and from volcanic emissions. However, excess Cl− can be present in some irrigated areas, usually as a result of high amounts of Cl in irrigation water or failure to apply sufficient water to adequately leach out Cl− accumulations.

Accumulation can occur under arid conditions.

Chlorine was established as an essential nutrient in 1954. Most plant species take up Cl− at relatively high rates. Soils considered low in Cl− contain water-soluble Cl in a concentration of less than 2 mg kg−1 of soil, which is rare (Mengel and Kirkby 2001). The beneficial effects of Cl− fertilization on plant growth are not fully understood, but improved plant-water relationships and inhibition of diseases are two important factors. In practice, Cl− deficiency very seldom occurs, because of the ubiquitous presence of Cl in the atmosphere and in rain and irrigation water, which is enough to meet the demands of crops. The effect of excess Cl− in plants is a more serious problem. Crops growing on salt-affected soils often show symptoms of Cl− toxicity.

Nickel Nickel (Ni) was established as an essential nutrient in higher plants in 1987. It is essential for plants supplied with urea. Ni-deficient plants accumulate toxic levels of urea in leaf tips, because of reduced urease activity. High Ni concentrations have toxic effects on plants. Most soils contain very small quantities of Ni, usually less than 100 µg g−1 of soil, well below the level at which Ni toxicity occurs. Long-term heavy applications of contaminated sewage sludge, how-

ever, may lead to a considerable rise in the Ni level in topsoil. Soils derived from ultrabasic igneous rocks, particularly serpentine, however, may contain 20 to 40 times this concentration, and Ni toxicity in plants is common in such soils (Mengel and Kirkby 2001).

Cobalt Cobalt (Co) is essential for microorganisms fixing N2, such as rhizobia, free-living N2− fixing bacteria, and blue-green algae. The total Co content in soils ranges from 1 to 70 mg kg−1 (Havlin et al. 2005). Cobalt occurs primarily in the crystal lattices of ferromagnesian minerals, and in this form it is unavailable to plants.

After release from these minerals by weathering, Co2+ is held largely in exchangeable form or as organomineral complexes. Exchangeable Co2+ is strongly bound, and its concentration in the soil solution, like that of Cu2+, is extremely low (Mengel and Kirkby 2001).

Cobalt is also bound to FeIII and MnIV oxides, and transient waterlogging conditions may lead to a reduction of FeIII and MnIV associated with a release of Co2+.

This is reflected in a higher concentration of available Co in poorly drained soils than in freely drained soils derived from the same parent materials (Mengel and Kirkby 2001). Cobalt availability is favored by increasing acidity and waterlogging. Soils in which Co deficiency can occur are acidic, highly leached sandy soils with low total Co, some highly calcareous soils, and some peaty soils (Havlin et al. 2005). Cobalt is adsorbed on the exchange complex and occurs in complexes of clay and organic matter. Fe, Al, and Mn oxides have a high adsorption capacity for Co and are capable of fixation of soil-applied Co fertilizer.

Beneficial Elements Some elements, while not proven to be essential for all plants, nevertheless appear beneficial for the growth and development of certain plants. The chemistry of one beneficial element in the soil is discussed here.

Silicon Silicon (Si) is the second most abundant element in the earth’s crust and is a major constituent of many soils (Datnoff et al. 2001; Ma and Takahashi 2002). However, it generally exists as a relatively insoluble component of primary minerals and secondary minerals (e.g., clays). Silicon is lost as soils weather, so highly weathered soils, such as those in the humid subtropics and tropics, usually contain less Si than soils in temperate

THE CHEMISTRY OF PLANT NUTRIENTS IN SOIL

regions. The concentration of Si in the soil solution may range from 1 to 40 mg L−1 and seems to be controlled more by chemical kinetics than by thermodynamics (Hallmark et al. 1982). Plants absorb Si from the soil solution in the form of monosilicic acid (H4SiO4), also called orthosilicic acid, which is chemically very active.

It can react with Al, Fe, and Mn to form slightly soluble silicates. Monosilicic acid can combine with heavy metals such as cadmium, lead, Zn, and mercury. The anion of monosilicic acid (Si(OH)3−) can replace phosphate anions (e.g., HPO42−) from Ca, Mg, Al, and Fe phosphates, thereby making P more readily available to plants. Silicon sorption in soils is pH-dependent, with greater sorption and therefore less availability to plants at higher pH. In addition to monosilicic acid, polymerized forms (polysilicic acids) are also an integral component of the soil solution. The mechanism of polysilicic acid formation is not clearly understood. It generally forms when Si in the soil solution exceeds 65 mg L−1.

Unlike monosilicic acid, polysilicic acid is chemically inert. It largely affects the physical properties of soil, acting as an adsorbent and forming colloidal particles.

Benefits attributable to Si have not been demonstrated for all crops, but substantial yield increases and pest resistance have been shown when Si fertilizer is applied to rice (Oryza sativa L.) and sugarcane (Saccharum sp.) grown in soils containing relatively little soluble Si.

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References Brady, N. C., and Weil, R. R. 2002. The Nature and Properties of Soils. 13th ed. Prentice Hall, Upper Saddle River, N.J. Datnoff, L. E., Snyder, G. H., and Korndörfer, G. H., eds. 2001.

Silicon in Agriculture. Elsevier Science, Amsterdam.

Hallmark, C. T., Wilding, L. P., and Smeck, N. E. 1982.

Silicon. Agron. Monogr. 9:263–265.

Havlin, J. L., Beaton, J. D., Tisdale, S. L., and Nelson, W. L. 2005. Soil Fertility and Fertilizers. 7th ed. Pearson Prentice Hall, Upper Saddle River, N.J. Lindsay, W. L. 1979. Chemical Equilibria in Soils. John Wiley & Sons, New York.

Ma, J. F., and Takahashi, E. 2002. Soil, Fertilizer, and Plant Silicon Research in Japan. Elsevier Science, Amsterdam.

Mengel, K., and Kirkby, E. A. 2001. Principles of Plant Nutrition. 5th ed. Kluwer Academic Publishers, Dordrecht, The Netherlands.

Norvell, W. A. 1972. Equilibria of metal chelates in soil solution. Pages 115–138 in: Micronutrients in Agriculture. J. J. Mortvedt, P. M. Giordano, and W. L. Lindsay, eds. Soil Science Society of America, Madison, Wisc.

Paul, E. A., and Clark, F. E. 1996. Soil Microbiology and Biochemistry. Academic Press, London.

Russell, E. W. 1961. Soil Conditions and Plant Growth. 9th ed. John Wiley & Sons, New York.

Snyder, G. H., Burt, E. O., and Gascho, G. J. 1979. Correcting pH-induced manganese deficiency in bermudagrass turf. Agron. J. 71:603–608.

2 The Physiological Role of Minerals in the Plant Ronald W. Rice University of Florida, Institute of Food and Agricultural Sciences Everglades Research and Education Center, Belle Glade

Introduction

manganese [Mn], zinc [Zn], copper [Cu], and molybdenum [Mo]).

Classifying essential nutrients based on levels deemed sufficient for growth (Table 2.1) may be somewhat misleading, since large variations exist across plant species and growing environments, and thus a number of essential elements (particularly micronutrients) may be present in plant tissues at levels far exceeding any physiological requirement.

Recognizing this limitation, Mengel and Kirkby (2001) offered a classification of nutrient elements by broad biochemical and physiological functions (Table 2.2).

Consistently with the evolving opinions shared by a growing cadre of plant physiologists, silicon (Si) and sodium (Na) are included in the list.

Under specific growing conditions and in a small number of plant species (mostly lower plants, such as algae), several additional elements occasionally receive a vote for essentiality, including cobalt (Co), vanadium (V), nickel (Ni), and selenium (Se). However, almost all plant investigators relegate these elements to the dubious category of â&#x20AC;&#x153;beneficial elements,â&#x20AC;? and many hasten to add Si and Na to this group. The discussion that follows presents examples of major physiological and metabolic roles played by the classic 16 elements (Table 2.1) in plant function and highlights Si as a worthy contender for essentiality.

Higher plants require a supply of certain inorganic elements in order to satisfy their nutritional requirements for growth and metabolic processes. Elements essential to plant growth are often called essential mineral elements, but, strictly speaking, they are not true minerals, although most (the exceptions are hydrogen, carbon, and oxygen) are ultimately derived from the breakdown products of larger mineral materials. The classic definition of essentiality, originally proposed by Arnon and Stout (1939), includes the following three criteria: 1. In the absence of an essential nutrient element, the plant fails to complete its full life cycle (from seed germination through the production of viable seed).

2. The essential function performed by the element is specific and cannot be duplicated by another nutrient element.

3. The element must be directly involved in plant metabolism (it is required for one or more specific physiological functions).

With strict adherence to these criteria, there are 16 widely recognized essential plant nutrients (Table 2.1), which are somewhat arbitrarily divided into nutrients typically required by plants in fairly great quantities, called macronutrients (hydrogen [H], carbon [C], oxygen [O], nitrogen [N], potassium [K], calcium [Ca], magnesium [Mg], phosphorus [P], and sulfur [S]), and those required in notably smaller quantities, called micronutrients (chlorine [Cl], boron [B], iron [Fe],

Hydrogen, Carbon, and Oxygen Hydrogen, carbon, and oxygen are distinct from other plant nutrients in several ways. First, these elements 9

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CHAPTER 2

are present in plants at levels far exceeding those of other nutrients (Table 2.1). Second, while other nutrients typically enter the plant as ionic, chelated, or small-molecular constituents of the soil solution, the vast portion of plant H, C, and O enters by decidedly different routes. The photosynthetic assimilation of atmospheric CO2, following its passage through leaf

stomata, serves as the primary source of plant C and a major source of plant O. Atmospheric O2 entering stomatal openings can also be metabolized by plants, a problematic situation when O2 directly competes with CO2 for ribulose-1,5-bisphosphate carboxylase/oxidase (rubisco) active sites. The bulk uptake and flow of molecular water from roots to shoots is driven by

Table 2.1. Approximate concentrations of mineral nutrient elements in plant dry matter (DM) deemed sufficient for adequate growtha Concentration in plant DM Element Micronutrients Nickel Molybdenum Cobalt Copper Zinc Sodium Manganese Boron Iron Chlorine Macronutrients Silicon Sulfur Phosphorus Magnesium Calcium Potassium Nitrogen Oxygen Carbon Hydrogen a

Chemical symbol

µmole g−1

mg kg−1

%

0.001 0.001 0.002 0.10 0.30 0.40 1.0 2.0 2.0 3.0

0.05 0.1 0.1 6 20 10 50 20 100 100

— — — — — — — — — —

Ni Mo Co Cu Zn Na Mn B Fe Cl Si S P Mg Ca K N O C H

30 30 60 80 125 250 1,000 30,000 40,000 60,000

— — — — — — — — — —

0.1 0.1 0.2 0.2 0.5 1.0 1.5 45 45 6

Number of atoms (relative to Ni) 1 1 2 100 300 400 1,000 2,000 2,000 3,000 30,000 30,000 60,000 80,000 125,000 250,000 1,000,000 30,000,000 40,000,000 60,000,000

Adapted from Epstein and Bloom 2005.

Table 2.2. Classification of plant nutrients based on predominant biochemical and physiological functionsa Nutrient elements

Uptake form

Biochemical and physiological functions

First group C, H, O, N, S

CO2, HCO3−, H2O, O2, NO3−, NH4+, N2, SO42−, SO2, as ions from the soil solution and as gases from the atmosphere

Major constituents of organic material; essential elements of atomic groups which are involved in enzymatic processes; assimilation by oxidationreduction reactions

Second group P, B, Si

Various phosphates, boric acid or borate, silicic acid; all from the soil solution

Esterification with native alcohol groups in plants; the phosphate esters are involved in energy transfer reactions

Third group K, Na, Ca, Mg, Mn, Cl

Ions from the soil solution

Nonspecific function establishing osmotic potentials; specific reactions in which the ion brings about optimum conformation of an enzyme (activation), bridging between reaction partners, charge-balancing anions, controlling membrane permeability and electrochemical potentials

Fourth group Fe, Cu, Zn, Mo

Ions or chelates from the soil solution

Present predominantly in a chelated form, incorporated with prosthetic groups that mediate catalytic reactions, undergo valency changes which mediate electron transport

a

Adapted from Mengel and Kirkby 2001.

THE PHYSIOLOGICAL ROLE OF MINERALS

the prevailing transpiration stream, and subsequent diffusion within all plant tissues distributes H and O throughout the plant. To a lesser degree, both H and O also enter plants from the soil solution as constituents of other inorganic nutrients (e.g., H2PO4−, NH4+, and SO42−) present in the uptake stream.

Various specific plant functions may be attributed to each of these three elements (for example, the role of H+ in proton pump mechanisms and the establishment of pH gradients across membranes that drive solute transport). However, it is the combination of all three inorganic elements into organic compounds that clearly defines the dominant function of H, C, and O in plants.

Organic sugars (carbohydrates) serve as metabolic substrates that drive a vast number of critically important plant physiological processes (glycolysis, etc.). The arrangement of H, C, and O into macromolecular organic constituents, such as cellulose, hemicellulose, and lignins, determines the basic structural components of all plant tissues. Organic molecules underlie the structure and function of amino acids, their proteins and enzyme systems, nucleic acids, and the entire genomic replication apparatus. In summary, all life depends on the organic composition determined by molecular associations of C and H and the tripartite configurations of organic acids composed of H, C, and O.

Nitrogen Unlike other essential plant nutrients, N can be taken up by plant roots either as an anion (nitrate, NO3−) or as a cation (ammonium, NH4+). Nitrate is not toxic to the plant, is readily translocated from the root apoplast through the xylem to shoots and leaves, and is safely sequestered at high concentrations in cell vacuoles.

However, further use of NO3− requires that it first be reduced to nitrite (NO2−) in the cell cytoplasm (a process involving a two-electron transfer mediated by the

11

enzyme nitrate reductase) and that the NO2− then be reduced to ammonia (NH3) in the chloroplast (a process involving a six-electron transfer from ferredoxin, mediated by the enzyme nitrite reductase) (Figure 2.1).

The NH3 is then readily assimilated into organic molecules by the glutamine synthetase−glutamate synthase pathway (Figure 2.2) (Beevers and Hageman 1983).

On the other hand, NH4+ (or its equilibrium species, NH3) is quite toxic at low concentrations. The deleterious accumulation of NH4+ in plant roots is avoided by the rapid incorporation of NH4+ into amino acids, notably glutamate (Figure 2.2), to form the N-rich amide glutamine. Subsequent glutamine deamination (loss of the amino [NH2] group) simultaneously regenerates the original glutamate (which may enter another NH4+ assimilation cycle) and produces a second glutamate when 2-oxoglutarate is aminated with the recently assimilated amino residue. This glutamate is readily translocated via the xylem to distal sites, where it can support the synthesis of essential N compounds.

The role of N in plant function is extensive. Nitrogen is an essential constituent of amino acids. Once N is incorporated into glutamate or glutamine, transaminase enzymes catalyze the formation of new amino acids by transferring the amino group to different carbon skeletons emanating from carbon fixation (Calvin cycle), glycolysis (breakdown of hexose sugars), and the tricarboxylic acid (TCA) cycle (Krebs cycle). Unlike humans, plants synthesize all transaminases needed to produce the 20 specific amino acids required for the synthesis of all required plant proteins.

Repeating sequences of peptide bonds between the carboxyl group of one amino acid and the amino group of another amino acid form the polypeptide chains that constitute the primary structure of all proteins.

Noncatalytic proteins serve a number of functions, including providing structural support (in microtubules, microfilaments, and organelle membranes), acting as electron carriers during photosynthesis and res-

Figure 2.1. Assimilation of nitrate (NO3−) across cytoplasmic and chloroplastic environments. FADH2 = reduced flavin adenine dinucleotide; Ferredox. = ferredoxin; ox. = oxidized; red. = reduced. (Reprinted from Marschner 1995, copyright 1995, with permission from Elsevier)

12 CHAPTER 2

piration (cytochromes), and acting as a seed reservoir of amino acids that can support seed germination and early seedling growth (Salisbury and Ross 1985).

However, the pervasive and indispensable essentiality of N as a plant nutrient is based primarily on its role as a component of enzymes, which are either entirely protein or largely composed of protein. Enzymes catalyze virtually every anabolic reaction (formation) and catabolic reaction (breakdown) within the plant and orchestrate the rate, timing, direction, and extent of most metabolic reaction pathways. Roughly 80 to 85% of the total N in green plant material is sequestered in protein. The remainder is distributed in nucleic acids (about 10%) and across the soluble metabolic pool of amino acids (about 5%) and their amines and amides (Mengel and Kirkby 2001).

Nucleic acids are linear polymers of nucleotides.

A nucleotide monomer is composed of a phosphorylated five-carbon sugar (ribose or deoxyribose) with an N-containing heterocyclic ring structure attached to the number 1 carbon of the pentose (Figure 2.3A). Five specific ring structures (bases), including two purines (adenine and guanine) (Figure 2.3B) and three pyrimidines (thymine, cytosine, and uracil) (Figure 2.3C), are essential to nucleic acid structure and function.

Deoxyribonucleotides are the monomers of DNA, and ribonucleotides are the structural units of RNA. As a structural component of the purines and pyrimidines that constitute nucleic acids, N functions at the very heart of the genetic code, within the cell nucleus during the synthesis (transcription) of messenger RNA (mRNA) from DNA and then in the cell cytoplasm where mRNA-coded proteins or enzymes (translation) are synthesized at the surface of ribosomes.

Adenosine triphosphate (ATP) is one of the aforementioned N-containing nucleotides (Figure 2.3D).

Upon hydrolysis and dephosphorylation (loss of one or two phosphate groups), ATP releases energy that subsequently fuels essential cellular activities, such as the active uptake of some essential inorganic nutrients and the transport of solutes and metabolites across otherwise impermeable cellular membranes. This process often creates pH gradients or electrochemical gradients across membranes, which in turn favor additional metabolic reactions. ATP also fuels starch synthesis and several critical steps in carbon fixation, including the regeneration of the CO2 acceptors ribulose1,5-bisphosphate in the C3 (Calvin cycle) pathway and phosphoenolpyruvate (PEP) in the C4 pathway.

Nicotinamide adenine dinucleotide (NAD+) and its phosphorylated form (NADP+) and reduced forms (NADH and NADPH) each carry two heterocyclic N ring structures. Collectively, these compounds act as electron donors or acceptors in many critically important metabolic pathways. For example, electrons accepted by ferredoxin at the terminal end of the photosynthetic electron transport chain (photosystem I [PS I] in chloroplast thylakoid membranes) are ultimately accepted

by NADP + (reduced to NADPH), which drives another electron transport cycle that ultimately splits water, evolves O2, and generates energy for ATP synthesis.

During the respiratory breakdown (glycolysis, TCA cycle) of hexose sugars, the repeated transfer of electrons from organic acid intermediates (pyruvate, isocitrate, Îą-ketoglutarate, L-malate) to NAD+ produces reduced NADH, which subsequently contributes the electrons required for ATP synthesis in the mitochondrial electron transport system. Various forms of NAD+ are also

Figure 2.2. Pathways for the assimilation of ammonia (NH3) (1 and 2), including the glutamine synthaseâ&#x2C6;&#x2019;glutamate synthase pathway with low NH3 supply (1) and high NH3 supply (2). The glutamate dehydrogenase pathway (3) is also described.

GOGAT = glutamine oxalate amino transferase. (Reprinted from Marschner 1995, copyright 1995, with permission from Elsevier)

THE PHYSIOLOGICAL ROLE OF MINERALS

involved in the reduction of NO3− to NO2− (Figure 2.1), fatty acid synthesis, and the conversion of fats to sugars.

Nitrogen is arguably essential to the entire photosynthetic apparatus, since it is contained in individual pyrrole subunits that form the tetrapyrrole ring structure common to all light-absorbing chlorophyll molecules (Figure 2.4A). The tetrapyrrole ring gives chlorophyll its green color, and the term chlorotic suggests an absence of green, a typical manifestation of N deficiency that reflects compromised chlorophyll function within chloroplasts. The N-containing tetrapyrrole ring is also contained in heme (Figure 2.4B), which functions as a prosthetic (nonprotein) group in several important enzyme systems (catalases, peroxidases, and cytochrome oxidases). Heme is also the central structural compo-

13

nent of the different cytochromes that participate in the electron transport systems in both mitochondrial membranes (where respiratory processes lead to ATP synthesis during oxidative phosphorylation) and thylakoid membranes (where light energy is captured by photosystem I [PS I] and photosystem II [PS II], water is split, O2 is evolved, and ATP is synthesized).

Phosphorus Phosphorus is taken up by plants as either HPO42− or H2PO4−, with the divalent anion dominating at elevated soil pH (>7.2). Active uptake mechanisms (H+ cotransport and/or HCO3− antiport) are required

Figure 2.3. Components of nucleotides underlying DNA and RNA structures: (A) the five-carbon pentose sugar, (B) purine N bases, and (C) pyrimidine N bases. (D) ATP structure, highlighting the role of Mg2+ in ATP function. (Redrawn from Mengel and Kirkby 2001)

14 CHAPTER 2

Figure 2.4. (A) Chlorophyll molecule and (B) related heme complex. (Redrawn from Mengel and Kirkby 2001)

to maintain cellular phosphorus at levels greatly exceeding those found in the soil solution.

Plant phosphorus is predominantly found in four different states: (1) as free inorganic orthophosphate (Pi), the form originally uptaken by roots; (2) as energy-rich pyrophosphate bonds (Pi units attached to one another), common to ATP (Figure 2.3D) and other nucleotide triphosphates; (3) as Pi attached by a single phosphate ester bond to a hydroxyl group of organic sugars or alcohols (i.e., fructose-6-phosphate, ribulose1,5-bisphosphate, and other intermediary phosphorylated metabolic compounds); and (4) as Pi forming diester bonds between organic molecules.

Pyrophosphate bond formation and degradation (hydrolysis) highlights the ubiquitous role phosphorus plays in the energy balance underlying major plant metabolic pathways. Energy absorbed during photosynthesis or released during respiration fuels the formation of energy-rich pyrophosphate bonds in ATP synthesis. Subsequent hydrolysis of these bonds drives the active transport of nutrients and solutes across otherwise impermeable membranes and releases energy required for enzyme activation, N2 fixation, and synthesis of organic compounds like starch. While ATP is arguably the most prolific nucleotide triphosphate, uridine, cytidine, and guanosine triphosphates are required for the synthesis of sucrose, phospholipids, and cellulose (cell wall component), respectively (Mengel and Kirkby 2001; Marschner 1995). Additionally, these nucleotides, together with deoxythymidine triphosphate, are the monomer units composing RNA and DNA structures.

The stepwise formation and degradation of phosphate ester bonds help drive the forward progress

of metabolic pathways. For example, the consecutive phosphorylation of intermediary sugar compounds in glycolysis (glucose → glucose-6-phosphate → fructose1,6-bisphosphate → etc.) essentially represent energy transfers, enabling newly phosphorylated compounds to undergo further metabolism.

The ability to form diester bonds between organic compounds highlights the structural role of P in several important plant compounds. Phosphate diester bonds link the sugar components of the five different nucleotides forming the linear chain configuration of RNA and DNA. Similarly, phosphorus plays a structural role in the various phospholipids that compose many cellular membranes by linking the glycerol backbone (with attached fatty acid chains) to an amino or alcohol group. The abundance of negatively charged oxygen atoms in phosphates confers hydrophilic membrane properties and also promotes stability across the membrane’s hydrophilic surface by interacting with cations, notably calcium (Figure 2.5).

As Pi (free inorganic orthophosphate), phosphorus has several regulatory functions, such as influencing activities of several key enzymes involved in carbohydrate metabolism and influencing photosynthate partitioning between starch and sucrose. Cytoplasmic Pi influences the activation of 6-phosphofructo-2-kinase, which catalyzes the synthesis of fructose-2,6-bisphosphate, a molecule that in turn activates the essential glycolytic enzyme phosphofructokinase (Stitt 1990). Elevated Pi in the stroma inhibits the enzymes responsible for catalyzing starch synthesis in chloroplasts (starch synthetase or ADP-glucose pyrophosphorylase) (Portis 1982).

Phosphorylated three-carbon sugars (3-phosphoglyceraldehyde and dihydroxyacetone phosphate) are

THE PHYSIOLOGICAL ROLE OF MINERALS

15

Figure 2.5. Schematic of the membrane bilayer, highlighting the hydrophilic region (left) and the hydrophobic region (right).

(Redrawn from Mengel and Kirkby 2001)

products of CO2 fixation in the Calvin cycle. An active antiport mechanism within the chloroplast membrane exports these triosephosphates into the cytoplasm in exchange for Pi entry into the stroma (Heber and Walker 1979). Low cytoplasmic Pi compromises the antiport mechanism, and increasing levels of triosephosphates in the stroma favor their incorporation into starch molecules. Conversely, elevated cytoplasmic Pi encourages triosephosphate export at the expense of starch accumulation. Cytoplasmic triosephosphates are subsequently used to synthesize many organic compounds, notably sucrose, cellulose (cell wall component), additional phosphorylated carbohydrate metabolites (fructose-6-phosphate, glucose1-phosphate), fatty acids, and amino acids.

Potassium Potassium is taken up by roots as the K+ cation.

Root ionophores (membrane-bound proteins that act as channels for specific cations) allow the passive diffusion of K+ into the root cell cytoplasm in response to the prevailing K+ concentration gradient. Active transport also mediates K+ uptake, whereby ATP activates a membrane-bound proton pump that ejects H+ from the cytoplasm, establishing an electrochemical gradient that favors the K+H+ symport (entry of both ions) back into the root cell cytoplasm (Maathuis and Sanders 1996).

Because the concentration of cytoplasmic K+ greatly exceeds that of any other cation, K+ plays a key role

in cellular osmotic function. In general, cellular water status (or turgor) is dictated by cellular K+ concentration (Läuchli and Pflüger 1978). The role of K+ as an osmoticum is highlighted by its influence on stomatal aperture.

Under illumination, ATP produced by photophosphorylation in stomatal guard cell chloroplasts fuels the active (proton pump) import of K+ (typically accompanied by charge-balancing organic acid anions, such as malate) from neighboring leaf epithelial cells into guard cell vacuoles. Declining osmotic potential within the guard cell favors the entry of water, and stomata are forced open as turgor increases (Humble and Raschke 1971).

Similar osmotic principles are responsible for photonastic movement (occurring in response to light) and thigmonastic movement (occurring in response to touch) of leaves of plants harboring specialized arrangements of “motor cells” (collectively, the pulvinus organ) at the base of petioles, blades, or leaflets. The external stimulus activates K+ transport (along with transport of Cl− or malate, or both) from one side of the pulvinus to the other. Motor cells gaining osmoticum swell, while those losing osmoticum shrink, and the resulting turgor differential forces leaf movement. A reversal in stimuli elicits a reversal in leaf movement (Satter and Galston 1981; Satter et al. 1988).

Potassium functions in a wide array of cell regulatory processes, activating at least 60 different enzymes in meristematic tissues (Suelter 1985) by inducing enzyme conformational changes that improve enzyme affinity for the substrate. In low-K+ regimes, impaired enzyme activities lead to declining plant starch reserves and an

16 CHAPTER 2

accumulation of soluble carbohydrates, reflecting the role K+ plays in the activation of starch synthetase, an enzyme that appends glucose monomers to growing starch molecules (Nitsos and Evans 1969), and critical glycolytic enzymes (phosphofructokinase, pyruvate kinase) (Läuchli and Pflüger 1978). Potassium also activates cell plasma membrane-bound proton-pumping ATPases (Fisher et al. 1970), triggering solute (including K+) transport through the membrane while also creating transmembrane pH gradients that help drive additional cellular metabolic processes. For example, in thylakoid membranes, K+ both activates ATPase and serves as the primary counter-ion (outward bound) to the light-triggered inward transport of H+ from the chloroplastic matrix (stroma) into the intrathylakoid space. The transmembrane pH gradient drives the photophosphorylation formation of ATP, while elevated pH and K+ concentrations in the stroma favor the synthesis and activity of rubisco, which consequently supports increased CO2 fixation in the Calvin cycle (Läuchli and Pflüger 1978; Peoples and Koch 1979).

Potassium also plays a role in meristematic cell growth by virtue of processes unleashed during ATPase activation. A loosening of the cell wall matrix combined with cell swelling supports the expansion of cell volume. Potassium activation of membrane proton pumps results in the outward transport of H+ from the cytoplasm, which increases cell wall extensibility by acidifying the apoplastic region. Meanwhile, ions and solutes are transported into the vacuole, which expands as water accumulates in response to declining osmotic potentials.

Carbohydrate partitioning is also influenced by K+ (Hartt 1969), which greatly facilitates the loading of sucrose from source leaves into phloem sieve tubes.

Again, the role of K+ and ATPase activation is implicated, whereby the established pH gradient drives a proposed H+-sucrose cotransport through the membrane into the sieve tube (Giaquinta 1977). The entry of K+ into the sieve elements also establishes an osmotic regime that helps drive the mass-flow movement of sucrose and other solutes to sink tissues (Marschner 1995).

Protein synthesis requires K+, which may help coordinate the binding of transfer RNA to ribosomal surfaces in the cytoplasm during the translation phase (Wyn Jones and Pollard 1983). At low levels of K+, plant protein declines, while soluble N compounds (amino acids, amides, NO3−, etc.) accumulate (Koch and Mengel 1974).

Sulfur Roots take up sulfur as the sulfate anion (SO42−) via an active H+ cotransport mechanism. Leaf stomata can also absorb atmospheric sulfur compounds, often in significant quantities near industrialized areas. The

assimilation of sulfur is initiated by ATP sulfurylase, an enzyme that exchanges the pyrophosphate (Pi−Pi) group of ATP for the sulfuryl group, producing adenosine phosphosulfate (APS). At this point, sulfur can follow a nonreduction assimilatory pathway (Figure 2.6), with the ATP-mediated conversion (phosphorylation) of APS to phosphoadenosine phosphosulfate (PAPS). The energy-rich PAPS-sulfate entity remains highly oxygenated and readily forms sulfate ester complexes (Schiff et al. 1993), notably those forming the sulfolipid compounds composing many cellular membranes, particularly chloroplastic thylakoid membranes. However, the synthesis of most S-bearing plant compounds requires SO42− reduction (ultimately, loss of oxygen atoms).

Following the formation of APS, the SO42− reduction pathway is mediated by the electron donor ferredoxin, and the reduced S-H entity is ultimately assimilated into the three-carbon amino acid cysteine (Figure 2.6).

Cysteine is precursor to the synthesis of all other reduced-S organic compounds, including the fourcarbon amino acid methionine, proteins and enzymes harboring cysteine or methionine, various coenzymes and prosthetic groups, and several vitamins. Aside from its constituent role in two amino acids, S also influences protein tertiary conformation (thus influencing enzyme function) through the formation of disulfide bonds between cysteine units located within the same polypeptide chain or across different polypeptide chains. For example, in plastocyanin, sulfur residues from cysteine and methionine coordinate with two histidine units to form the “cagelike” binding site for Cu2+ (Figure 2.7), which ultimately mediates the electron transfer role of plastocyanin in the photosynthetic electron transfer chain (Gross 1996).

Sulfur profoundly influences a wide variety of plant metabolic processes by influencing the structure and enzymatic function of nonheme Fe-S proteins, notably ferredoxin. Multiple cysteine S residues in ferredoxin polypeptide chains coordinate with Fe and additional S atoms, forming a highly reactive redox cluster that readily accepts and donates electrons. During photosynthesis, ferredoxin participates in the electron transport system within thylakoid membranes (i.e., O2 generation and photophosphorylation synthesis of ATP), and in the chloroplastic stroma it donates electrons to NADP+ (Figure 2.7), producing the NADPH required to fix CO2 in both the C3 and C4 fixation pathways. Ferredoxin mediates the assimilation of the plant nutrients NO3− (Figure 2.1) and SO42− (Figure 2.6) and initiates the incorporation of reduced N (Figure 2.2) into organic compounds. It also initiates the biological reduction of atmospheric N2 by supplying electrons to nitrogenase. These electrons stimulate additional Fe-S redox clusters within the nitrogenase enzyme complex, producing a highly energized electron transfer site where N2 fixation can take place. Sulfur functions similarly in numerous other enzyme systems harboring

THE PHYSIOLOGICAL ROLE OF MINERALS

Fe-S cofactors, notably glutamate synthetase, which is involved in NH3 assimilation via the glutamine oxalate amino transferase (GOGAT) pathway (Figure 2.2); ubiquinone reductase, which is involved in electron transfer within mitochondrial membranes; and aconitase, which converts citrate to isocitrate in the TCA cycle.

Sulfur is a structural component of thiamine (vitamin B1) and biotin (vitamin H). Vitamin B1 is the precursor of thiamine pyrophosphate, which functions in tandem with additional S-containing cofactors (Îą-lipoic acid and coenzyme A) in an enzyme complex that catalyzes the irreversible conversion of pyruvate to acetyl coenzyme A (acetyl-CoA) (Figure 2.8).

Sulfur-containing biotin serves as a cofactor for en-

17

zymes that catalyze the transfer of carboxyl groups, notably in fatty acid synthesis and the initiating step of glucose synthesis, when the three-carbon pyruvate (or PEP) is converted into the four-carbon oxaloacetate.

Reduced sulfur functional groups, termed sulfhydryl groups (S-H), participate in many metabolic processes, by means of their electron-donating properties.

The reactive redox centers of Fe-S proteins (discussed above) are actually coordinated between sulfhydryl entities on polypeptide cysteine units. The terminal cysteine sulfhydryl group on coenzyme A (CoA-SH) readily forms high-energy thioester bonds with acetic acid, forming acetyl-CoA. By donating energized acyl (two-carbon) groups, acetyl-CoA initiates the TCA

Figure 2.6. Sulfur assimilation pathways in higher plants and green algae, including (1) the synthesis of sulfate diesters and (2) sulfate reduction via the adenosine phosphosulfate (APS) pathway. Ferredox. = ferredoxin; ox. = oxidized; red. = reduced; PAPS = phosphoadenosine phosphosulfate. (Reprinted from Marschner 1995, copyright 1995, with permission from Elsevier)

Figure 2.7. Photosynthetic electron transport system in photosystem I (PS I) and photosystem II (PS II). Chl = chlorophyll; Cyt = cytochrome; Q = quencher; X = unknown compound; XAN = xanthophyll cycle. (Reprinted from Marschner 1995, copyright 1995, with permission from Elsevier)

18

CHAPTER 2

Figure 2.8. Role of sulfur-bearing compoundsâ&#x20AC;&#x201D;thiamine pyrophosphate (TPP), sulfhydryl-disulfide redox system of lipoic acid, and the sulfhydryl group of coenzyme A (CoA-SH)â&#x20AC;&#x201D;in the formation of acetyl-CoA, which subsequently participates in fatty acid synthesis and the Krebs cycle. (Reprinted from Marschner 1995, copyright 1995, with permission from Elsevier)

Figure 2.9. A, Glutathione redox system in the HalliwellAsada pathway. Glutath. = glutathione; oxid. = oxidized; red. = reduced. B, Role of superoxide dismutases, peroxidase, and catalase in the quenching of tissue-damaging reactive oxygen species. (Redrawn from Mengel and Kirkby 2001)

cycle and also provides the carbon building blocks for long-chain fatty acid synthesis (Figure 2.8).

Cysteine sulfhydryl units on various thioredoxin proteins readily reduce disulfide bonds in target proteins, enabling thioredoxin to activate enzymes operating in photosynthesis and carbon fixation and to mobilize starch and protein reserves during seed germination (Kobrehel et al. 1992; Besse and Buchanan 1997). Glutathione (a tripeptide of cysteine, glycine, and glutamate) plays several roles in plant defense systems. Environmental stresses (excessive light intensity, drought, chilling, wounding, nutrient deficiency, and foreign chemicals, such as ozone) can create imbalances in electron transport systems, generating an excessive supply of electrons, which favor the formation of toxic reactive oxygen species, such as the superoxide radical, hydroxyl radical, and hydrogen peroxide (H2O2), compounds that disrupt metabolic processes and damage cellular membranes, enzymes, and nucleic acids. Glutathioneâ&#x20AC;&#x2122;s sulfhydryl residue confers antioxidant properties, and by readily accepting electrons in the Halliwell-Asada pathway, glutathione participates in the detoxification of reactive oxy-

gen species (Figure 2.9A). Glutathione is also a building block of phytochelatins, proteins with repetitive glutamate-cysteine units, which protect plants against heavy metal damage by chelation via multiple sulfur bonds.

Finally, the distinctive flavor and pungency of some plant species are largely due to S-bearing compounds.

Following tissue damage in many plants in the Brassicaceae (Cruciferae), such as horseradish, turnip, cabbage, broccoli, and kale, enzymes released from disrupted cell vacuoles degrade glucosinolates into volatile S compounds (thiocyanates, isothiocynates), which impart recognizable odors. Similarly, in the genus Allium, the odorless precursor alliin is enzymatically converted to allicin (a thiosulfinate), which imparts the familiar redolence and flavor to garlic. A slightly different alliin and thiosulfinate chemistry confers the lachrymatory effect (bringing tears to the eyes) in other Allium species, notably onions, leeks, and chives. The volatile by-products emanating from glucosinolate and alliin degradation may confer some measure of plant defense against insects and fungal pathogens.

Calcium Calcium is absorbed as the divalent cation Ca2+. While other macronutrients can be found in appreciable quantities throughout the symplast, plant calcium is largely located in the apoplastic cell wall region.

Cytoplasmic concentrations of free Ca2+ are also very low, a result of mechanisms that sequester Ca2+ within cell organelles (vacuole, endoplasmic reticulum, and chloroplast). Proper metabolic function requires the compartmentalization of free Ca2+ in order to avoid precipitation reactions with inorganic phosphorus species (Pi), the formation of Ca salts with ATP and other organic phosphates, and competition for enzymebinding sites preferably reserved for Mg2+ and in order to allow the effective use of Ca2+ as a second messenger (Marschner 1995; Salisbury and Ross 1985).

Calcium plays a key role in several plant structures.

Cell wall stability and plant structural strength is predicated on the formation of Ca-pectate compounds, which stabilize the cellulitic matrix of the middle

THE PHYSIOLOGICAL ROLE OF MINERALS

lamella and “cement” adjacent plant cells to one another.

Tissue Ca2+ deficiencies promote increased activity of polygalacturonase, which enzymatically breaks down the cell wall pectate matrix, leading to either detrimental breakdown of plant tissues or beneficial ripening of tissues in fruits such as tomato (Rigney and Wills 1981).

Calcium also stabilizes the bilayer structure of phospholipid membranes by bridging phosphate and anion complexes located at the hydrophilic membrane surface (Figure 2.5). Calcium deficiencies can compromise membrane function by allowing leakage of inorganic and organic solutes across the lipid bilayers, which subsequently compromises metabolic processes.

Cell growth or extension and division require a general degradation of the Ca-pectate cell wall material, a growth process triggered by auxin, which mediates the acidification of the cell wall region (Cleland et al.

1990). However, auxin also activates the entry of free Ca2+ into the cytoplasm, which initiates the formation of organic compounds necessary for the synthesis of new cell wall material (Brummell and Hall 1987). Thus, Ca2+ plays a central role in the formation of new cells that sustain new growth, such as coleoptile and shoot elongation. The secretion of compounds that direct pollen tube growth is chemotropically directed along a Ca2+ gradient (Mascarenhas and Machlis 1964). Calcium is also required for the formation of the microtubules underlying the mitotic spindle apparatus during cell division (Salisbury and Ross 1985). Calcium regulates additional secretory processes, including the formation of mucilage that lubricates root cap extension through the soil and the synthesis of callose, which is deposited in response to mechanical or pest injuries.

Calcium directly activates a few enzymes, including several involved in cell membrane synthesis (Mengel and Kirkby 2001), Ca-dependent protein kinases (which phosphorylate additional enzymes, like the membranebound H+-pumping ATPases) (Roberts and Harmon 1992), and α-amylase (which mediates the degradation of stored starch into soluble glucose in leaf chloroplasts and in germinating seeds) (Salisbury and Ross 1985). However, Ca2+ is more prominently recognized by its action as a “second messenger”: environmental signals received at the cell surface are relayed by an influx of free cytoplasmic Ca2+, which targets specific Ca-binding proteins, notably calmodulin (Roberts and Harmon 1992). Upon activation by conformational change induced by Ca2+, calmodulin has been implicated in the allosteric activation of additional enzymes, such as NAD kinase (which converts NAD+ to NADP+, the terminal electron acceptor in the chloroplastic thylakoid membrane system), membranebound Ca-ATPase (which regulates the level of free cytoplasmic Ca2+), adenylate cyclase, and cyclic nucleotide phosphodiesterase (Zielinski 1998; Mengel and Kirkby 2001).

19

Magnesium Magnesium is taken up by plant roots as the divalent cation Mg2+. Magnesium’s role in plant structure is fairly limited, although it occupies a central coordinating position in the tetrapyrrole ring of chlorophyll molecules (Figure 2.4A). In contrast to Ca2+, Mg2+ is plentiful throughout the symplast, where it influences a vast number of metabolic processes by complexing with anionic molecules and with negatively charged ligands.

In many cases, Mg2+ induces conformational changes that favor accelerated enzyme activities and enhanced enzyme affinities for the substrate. For example, by forming ionic bonds across different components of ATP, Mg2+ induces the tertiary configuration required to bridge the ATP complex with the target enzyme that ultimately catalyzes the phosphorylation of the target molecule (Figure 2.3D). In the reverse scenario, various phosphatase enzymes require Mg2+ activation to catalyze dephosphorylation reactions. The synthesis of ATP also requires Mg2+, which ionically bridges ADP with the phosphorylating enzyme. The activity of many membrane-bound H+-pumping ATPases requires activation by an Mg2+-ATP complex rather than free ATP (Mengel and Kirkby 2001). A notable example includes ATPases in sieve tube membranes, which mediate sucrose loading into the phloem.

Photosynthetic light reactions result in the countertransport of Mg2+ and H+ across the thylakoid membrane, producing elevated pH and Mg2+ levels in the stroma, which activate fructose-1,6-bisphosphatase activity in the Calvin cycle. Furthermore, the same stroma conditions greatly enhance carbon fixation by rubisco. Again, the bridging coordination between Mg2+ and rubisco increases the enzyme’s affinity for CO2 (Pierce 1986). In C4 and Crassulacean acid metabolism (CAM) plants, the initial CO2 fixation event by PEP carboxylase requires Mg2+ activation. Other notable Mg2+activated enzymes include glutathione synthetase (which generates the S-rich tripeptide that quenches reactive oxygen species) and glutamine synthetase (which initiates NH3 assimilation into organic structures) (Figure 2.2).

Finally, the formation of proteins requires Mg2+, which activates RNA polymerase and bridges ribosome units into configurations that favor polypeptide synthesis.

Iron A large fraction of soil iron is present in insoluble forms. Although plant roots selectively uptake the Fe2+ cation, chelated Fe3+ forms predominate across most soil pH and aeration environments. In plants other than grasses, physiological mechanisms operating at root tips mediate the reduction of Fe3+ chelates, releasing the Fe2+ cation, which ultimately passes through the cell

20 CHAPTER 2

membrane into the cytoplasm (Fox and Guerinot 1998).

Using a different strategy, grasses synthesize and release low-molecular-weight siderophores, which chelate Fe3+ into a complex that is then taken up by roots. Because free iron is highly reactive, chelated forms are once again involved in long-distance transport in the xylem and phloem. Furthermore, iron is sequestered within organelles (notably chloroplasts) as ferritin (a protein shell surrounding up to 5,000 Fe atoms), which releases Fe in response to metabolic needs (Sechbach 1982).

The essential roles of iron in plant function are related to its ability to undergo reversible redox transtions between Fe3+ and Fe2+ and its readiness to participate in electron transfer reactions. Iron occupies the central position of the heme complex (Figure 2.4B), which functions as a prosthetic group in cytochromes.

By reversibly altering oxidation states in heme, iron can accept and then release electrons in photosynthetic (thylakoid membranes) and respiration (mitochondrial membranes) electron transport systems. The Fecontaining heme is also present in catalase and peroxidase enzyme systems. By catalyzing the breakdown of H2O2, both catalase and ascorbate peroxidase play leading roles in the detoxification of reactive oxygen species (Figure 2.9B). Other peroxidases react with H2O2 at cell wall surfaces to mediate the polymerization of phenolic compounds into cell wall lignins and suberin, the latter functioning as a protective coating along root surfaces (Casparian strip) and plant wound sites.

Iron dictates the enzymatic function of nonheme Fe-S proteins (ferredoxin, nitrogenase, aconitase, etc.), which were highlighted earlier (see Sulfur). Briefly, these enzymes contain redox-reactive Fe-S clusters that are coordinated with cysteine S residues on the polypeptide. By undergoing rapid and reversible valency changes, the iron constituent accepts or transfers electrons that ultimately catalyze the required reaction. Nitrogenase deserves additional mention, since it is actually a complex of two Fe-containing metalloproteins, generally termed the Fe protein (which contains a four-Fe−four-S cluster) and the MoFe protein. With N2 (N≡N) bound to the nitrogenase complex, the Fe protein accepts electrons from ferredoxin (or flavodoxin) and then binds into a complex with ATP that mediates electron transfer to the MoFe protein, which subsequently donates electrons to reduce N2 to HN=NH. A second electron transfer (reduction) cycle produces H2N−NH2, and a third generates the final N2 fixation product, 2NH3.

The various superoxide dismutases (SODs) are metalloenyzmes that initiate the catalytic breakdown of harmful superoxide radicals (Figure 2.9B), which can disrupt proteins, membranes, and DNA. The Fe-SOD isoenzyme predominates in chloroplasts (Bowler et al.

1992) and, in tandem with catalase (which detoxifies hydrogen peroxide), functions as a plant defense

mechanism during unbalanced photosynthetic electron supply. The formation of ethylene from methionine involves electron transfer mediated by an Fe enzyme. The various lipoxygenase enzymes all contain Fe, and during seed germination, rapid tissue growth, and tissue senescence these enzymes function to break down the fatty acid lipid constituents that compose cell membranes (Hildebrand 1989). Both Fe and S (cysteine residues) are activating components of ribonucleotide reductase, which converts ribonucleotides to deoxyribonucleotides, and hence is a key enzyme in the synthesis and repair of DNA (Reichard 1993). Iron-containing enzymes operate in the biosynthesis pathway, which forms the tetrapyrrole ring structure common to both heme and chlorophyll (Figure 2.4). Diverging metabolic pathways that ultimately synthesize heme and chlorophyll also require iron. Finally, numerous Fecontaining constituents (cytochromes, Fe-S proteins, etc.) of chloroplastic thylakoid membranes (Figure 2.7) participate in the electron transfer mechanisms of PS I and PS II (Terry and Abadía 1986).

Copper The mechanism of copper uptake is not well understood. Initial uptake events likely involve Cu2+ or a related chelation complex. Because of its highly reactive properties, the long-distance transport of Cu2+ in the xylem likely occurs as a complexed form with soluble N compounds, such as amino acids (Loneragan 1981). The numerous roles of copper in plant physiological processes are related to its role in enzymatic function. Copper shares several characteristics with iron, notably a readiness to undergo valency changes between Cu2+ and Cu+ and a propensity to participate in reactions requiring electron transfer.

Over half of the copper present in chloroplasts is bound to plastocyanin, a small protein bound loosely in thylakoid membranes. The Cu2+ atom in plastocyanin catalyzes a single-electron transfer linking PS II with PS I in the photosynthetic electron transfer chain (Figure 2.7). The CuZn-SOD isoenzyme, abundant in chloroplasts (Bowler et al. 1992), contains two copper atoms, which protect the photosynthetic apparatus by transferring electrons to cell-damaging superoxide radicals, converting them into H2O2 (Figure 2.9B). The CuZn-SOD performs the same function in mitochondria, thereby protecting the respiratory electron transport mechanism from oxidative damage during periods of unbalanced electron transfer.

As a component of cytochrome and Fe-S proteins, Fe clearly plays a pivotal role in the sequential transfer of electrons in mitochondrial inner membranes. However, the final electron transfer event completing the oxidative phosphorylation synthesis of ATP requires cytochrome

THE PHYSIOLOGICAL ROLE OF MINERALS

oxidase, an enzyme complex containing two copper and two heme-iron constituents, which collectively transfer the four electrons required to reduce O2 to H2O. Electrons transferred by copper determine the catalytic function of various phenol oxidases (or phenolases) that react with O2 to catalyze the synthesis of aromatic hydrocarbons. Cu-bearing monophenolase (or tyrosinase) converts monophenol amino acid tyrosine to diphenol dihydroxyphenylalanine, which can subsequently be converted into quinone by Cu-bearing polyphenolase.

The accumulation of quinones initiates the synthesis of brown melanin compounds, which form across plant wound surfaces (notably in apples, bananas, and potatoes). The nomenclature and biochemistry underlying phenolic transformations is beyond the scope of this discussion. In brief, Cu-bearing phenolases direct the synthesis of aromatic compounds required for the synthesis of lignin, an essential cell wall constituent, which confers structural strength to the middle lamella and xylem wall elements. The lignin matrix resists compression forces (e.g., in tree bark) and gives rigidity to xylem walls, allowing for the long-distance transport of water and nutrients under tension. Next to cellulose, lignin is the most abundant organic compound on earth (Salisbury and Ross 1985).

The multicopper enzyme ascorbate oxidase mediates a four-electron transfer in the conversion of ascorbic acid to dehydroascorbic acid, the initializing step in the Halliwell-Asada pathway, which neutralizes H2O2, thus finalizing the detoxification of harmful superoxide compounds (see Sulfur; Figure 2.9A) (Bowler et al. 1992; Marschner 1995). Finally, Cu-bearing diamine oxidase reacts with apoplastic polyamines to form H2O2, which is subsequently used by cell wall peroxidases for cell wall strengthening (lignification) and suberization responses to mechanical plant injuries (Scalet et al. 1991).

Zinc It is unclear whether plant uptake of divalent Zn2+ is mediated by ion channels or by membrane-bound ATP-activated proton pumps (Fox and Guerinot 1998).

Long-distance transport in the xylem involves both free Zn2+ and Zn bound to soluble organic solutes (Marschner 1995). In contrast to copper and iron, Zn2+ is stable in biological mediums and does not undergo valency changes, and hence zinc does not participate in electron transfer reactions (Vallee and Falchuk 1993). Nonetheless, the number of enzymes catalyzed by zinc (over 300) (Fox and Guerinot 1998) is far greater than the number catalyzed by other metals. A unique ability to form stable associations with polypeptide chains through multiple coordination numbers and geometries (ranging from regular and distorted tetrahedral to pyramidal and octahedral) en-

21

ables zinc to interact with a broad range of enzymes and proteins that collectively carry out many diverse biological functions (Vallee and Falchuk 1993).

The zinc component of carbonic anhydrase (an enzyme present in the cytoplasm and chloroplasts) plays a direct role in the catalytic interconversion between CO2 and HCO3â&#x2C6;&#x2019; (Marschner 1995). In this capacity, zinc plays an essential role in carbon fixation in C4 plants, supplying HCO3â&#x2C6;&#x2019; substrate for cytoplasmic PEP carboxylase and then CO2 substrate for rubisco (C3 pathway) in the chloroplastic stroma. As a component of both aldolase and fructose-1,6-bisphosphatase (Marschner 1995), zinc plays additional roles in carbon fixation. In the Calvin cycle, aldolase catalyzes the union of two triosephosphates (3-phosphoglyceraldehyde and dihydroxyacetone phosphate) to form fructose-1,6-bisphosphate.

This six-carbon compound is subsequently dephosphorylated by fructose-1,6-bisphosphatase to form fructose-6-phosphate. Both reactions underlie the cycling of CO2 fixation products that ultimately regenerate the CO2 acceptor ribulose-1,5-bisphosphate.

These two zinc-containing enzymes also influence carbohydrate metabolism. Repeated CO2 fixation cycles produce a surfeit of triosephosphates. Chloroplastic aldolase activity regulates the fate of these three-carbon compounds, namely, whether they are exported to the cytoplasm or accumulate as starch within chloroplasts.

In the cytoplasm, aldolase regulates a key step in glycolysis by splitting fructose-1,6-bisphosphate into two triosephosphates that ultimately generate pyruvic acid for the Krebs cycle. Cytoplasmic triosephosphates are also used to synthesize sucrose and polysaccharides (see Phosphorus). Additionally, fructose-1,6-bisphosphatase in the cytoplasm converts fructose-1,6-bisphosphate into fructose-6-phosphate, which also serves as a precursor for sucrose and polysaccharide formation.

In O2-limiting environments (waterlogged roots, etc.), several zinc-dependent enzymes indirectly influence the flow of glycolytic metabolites. In contrast to glycolysis, anaerobic conditions quickly compromise the Krebs cycle and mitochondrial electron transport system. Under these conditions, the accumulation of pyruvic acid and subsequent shutdown of glycolysis is prevented by fermentation. Two zinc atoms perform catalytic and structural roles in alcohol dehydrogenase (Coleman 1992), which mediates the conversion of pyruvic acid to ethanol. Another fermentation pathway involves the conversion of pyruvic acid to lactic acid by zinc-dependent lactic acid dehydrogenase. Both fermentation processes support the continuation of glycolysis under low-O2 conditions.

Zinc is a component of CuZn-SOD, one of several superoxide dismutase isoenzymes (see Copper; Iron; Manganese; Figure 2.9B) that protect cell membranes, proteins, and DNA from oxidative damage when unbalanced electron transfer occurs during photosynthesis and

22 CHAPTER 2

mitochondrial electron transport. Elevated levels of zinc and SOD enzymes may offer protection against sunscald damage in fruit and vegetable plants grown in environments with high temperatures and high light intensity (Marschner and Cakmak 1989; Bowler et al. 1992).

Other Zn-containing enzymes include carboxypeptidase (which catalyzes the hydrolysis of the C-terminal peptide bond, thus cleaving the C-terminal amino acid from the polypeptide chain), alkaline phosphatase (which hydrolyzes many different esters of phosphoric acid), and phospholipase (which catalyzes the hydrolytic cleavage of different fatty acid components) (Marschner 1995).

Zinc atoms in specific DNA-binding metalloproteins play a leading structural role underlying DNA replication, transcription of complementary RNA products from DNA, and translation (protein synthesis) of RNA information at ribosome surfaces (Coleman 1992; Vallee and Falchuk 1993). Briefly, the zinc atom ionically coordinates in tetrahedral fashion with cysteine or histidine residues present at different locations along the polypeptide chain, creating a series of loops, or “zinc finger motifs,” along the chain. This zinc finger conformation ensures correct binding to gene sequences on DNA (or RNA) and subsequently coordinates transcription and translation processes by directing the sequential placement of required amino acids to produce the desired protein end product. Additionally, zinc plays a similar structural role in RNA polymerase, inducing conformational structures within subunits that allow the enzyme to catalyze transcription. Zinc may also help stabilize ribosome structure and integrity, which would support the translation process (Prask and Plocke 1971).

Manganese Manganese is taken up as divalent Mn2+ and is transported through the xylem either as free Mn2+ or in weak association with soluble organic acids. Manganese plays a role in numerous plant redox processes by its ability to undergo changes in oxidation status between Mn2+ and Mn4+. Although Mn2+ can physically substitute for Mg2+ in many enzyme activation systems, the frequency of this substitution is often minimized by high Mg2+ concentrations in plant cell environments.

There are two widely recognized Mn-containing enzymes. The first is the MN-SOD isoenzyme (see Copper; Iron; Zinc; Figure 2.9B), which operates predominantly within mitochondria to quench tissuedamaging oxygen radicals (Bowler et al. 1992). The second is an Mn-protein complex found in the thylakoid membranes of photosystem II (Figure 2.7). Absorption of photon energy induces electron transfer from the P680 reaction center of PS II. Electrons lost from P680 are subsequently replaced by electrons donated from four Mn atoms clustered in the Mn protein.

The resulting oxidized status of the Mn cluster provides the driving force for the Hill reaction, namely, the oxidation (splitting) of two water molecules and the evolution of molecular oxygen (Figure 2.7).

As a cofactor, Mn2+ activates at least 36 enzyme systems (Burnell 1988). In this capacity, Mn2+ shares a role (with Mg2+) in several Krebs cycle processes, including the activation of NAD+-linked malate dehydrogenase (which oxidizes malic acid to oxaloacetic acid) and isocitrate dehydrogenase (which first converts isocitric acid to unstable oxalosuccinic acid and then converts the unstable intermediate to α-ketoglutaric acid) (Marschner 1995).

Manganese plays several critical roles in CO2 fixation in C4 plants. The assimilation of HCO3− by PEP in C4 mesophyll cells forms oxaloacetic acid, in a reaction catalyzed by PEP carboxylase and requiring Mn2+ or Mg2+ for activation. Depending on the carbon shuttle strategy, oxaloacetic acid is then preferentially reduced to malic acid (malate formers) or transaminated into aspartic acid (aspartate formers). Following the transfer of malic acid from the mesophyll into bundle sheath cells, decarboxylation (release of CO2 for the C3 cycle) is mediated by NAD+-malic enzyme, which has an absolute requirement for Mn2+ (Burnell 1988).

Depending on the plant species, the decarboxylation that initiates the C3 cycle in aspartate formers can be mediated by two different mechanisms, involving the activity of either NADP-malic enzyme (using Mn2+ or Mg2+) (Burnell 1988) or PEP carboxykinase (with an absolute requirement for Mn2+) (Burnell 1986).

A number of enzymes catalyzing the biosynthesis of aromatic compounds are activated by Mn2+. The shikimic acid pathway includes a series of reactions that give rise to the aromatic amino acids phenylalanine, tyrosine, and tryptophan. The pathway is initiated with the condensation of PEP and erythrose-4-phosphate to form the seven-carbon compound 1-carboxy-2deoxy-α-D-glucose-6-phosphate (DAHP), catalyzed by various DAHP synthases with catalytic properties that are either enhanced or directly activated by Mn2+ (Herrmann and Weaver 1999). Phenolic compounds formed in the pathway also serve as precursors for the biosynthesis of many additional aromatic compounds, including lignins, alkaloids, flavonoids, and related pigments. For example, upon activation by Mn2+, phenylalanine ammonia lyase catalyzes the deamination of phenylalanine to form cinnamic acid, which can initiate pathways leading to lignin and flavonoid products. The synthesis of gibberellic acid and various chloroplast pigments from isoprenoid precursors are catalyzed by enzymes that are either enhanced or directly activated by Mn2+ (Wilkinson and Ohki 1988).

In some tropical legumes, N-rich ureides (allantoin and allantoate) are the primary compounds in which N is transported from nodules, and subsequent

THE PHYSIOLOGICAL ROLE OF MINERALS

breakdown of these ureides (releasing NH3 for plant N metabolism) is catalyzed by allantoate amidohydrolase, which requires Mn2+ (Winkler et al. 1985). The transcription of chloroplastic DNA is directed by RNA polymerase and uses Mn2+ for activation at much lower concentrations than Mg2+ (Ness and Woolhouse 1980). Manganese also activates indoleacetic acid (IAA) oxidase, which impacts growth and development processes by catalyzing the oxidative degradation of IAA. Manganese is also involved in fatty acid synthesis, specifically activating the S-containing coenzyme biotin, which directs the transfer of carbon groups to the growing chains of fatty acids (Figure 2.8) (Marschner 1995).

Boron Roots take up B mainly as undissociated boric acid (H3BO3). Despite many investigative efforts, compelling evidence supporting the active uptake of B remains elusive. Hu and Brown (1997) suggested that B enters roots by passive diffusion, driven by the prevailing transpiration stream and favorable concentration gradients established by the rapid formation of B complexes in the cytoplasm and cell wall, which limits the concentration of free boric acid in the plant.

With respect to specific roles and functions played by essential plant nutrients in plant physiological processes, B has the distinction of being the least understood. Many investigations have described various anatomical, physiological, and biochemical

23

changes that rapidly develop in plants following the imposition of low-B environments and following the resupply of B to deficient plants. However, speculations regarding the role of B in plant function have been often contradictory, reflecting difficulties that underlie the distinction between primary and secondary effects of B nutrition on plant function (Marschner 1995).

Abnormal structural changes within cell walls have been linked to B deficiency. The cell wall environment harbors up to 90% of cellular B content (Loomis and Durst 1992). Boron provides structural linkages within cell walls (Figure 2.10) by readily forming diester complexes with hydroxyl groups of cell wall diols and polyols to form pectic polysaccharide complexes (Matoh 1997). Structural characteristics conferred by these B cis-diol cross-linkages between cell wall components could explain many of the visual symptoms observed in plants with B deficiency and toxicity (Blevins and Lukaszewski 1998).

Boron deficiencies are also associated with compromised cell membrane function, such as high rates of ion and solute leakage across the plasmalemma and disrupted activities of membrane-bound protonpumping ATPases. Cakmak et al. (1995) suggested that by forming H-bonding and cis-diol associations between various membrane glycoproteins and/or glycolipids, B serves to stabilize membrane structure, which in turn maintains membrane channels and membrane-bound enzymes in physiologically favorable conformations. Furthermore, by forming complexes with phenolic compounds, B likely provides cell membranes protection from damaging toxic quinones

Figure 2.10. Proposed roles of boron in cell wall metabolism, highlighting primary and secondary (cascade) effects that develop with boron deficiency. IAA = indoleacetic acid. (Reprinted from Marschner 1995, copyright 1995, with permission from Elsevier)

24 CHAPTER 2

and reactive oxygen species that arise when phenolics are oxidized (Cakmak et al. 1995). Several enzymes (e.g., IAA oxidase, ribonuclease, and polyphenoloxidase) that normally reside latently on cell membranes and walls are released in B deficiency and in turn can influence IAA levels (compromising tissue differentiation and growth processes), compromise RNA levels (compromising protein synthesis), and encourage the accumulation of phenolics (compromising lignin synthesis) and highly reactive tissue-damaging aromatic intermediates, such as quinones (Cakmak and Römheld 1997). However, Marschner (1995) suggested that these kinds of responses are secondary effects that cascade in response to the primary effects of B deficiency, namely, changes in cell wall structure and compromised cell membrane function (Figure 2.10).

Marschner (1995) noted that different experimental conditions have generated a number of seemingly contradictory relationships between B status, IAA, and phenolic metabolism. An accumulation of phenolic compounds along with their related enzymes are typical manifestations of B deficiency, likely initiated with the degradation of cell wall material. Additionally, under low-B conditions, substrate flux is shifted from glycolysis towards the pentose phosphate pathway, which generates the intermediate erythrose-4-phosphate, one of two essential compounds that initiate the synthesis of many phenolic compounds via the shikimic acid pathway (Mengel and Kirkby 2001).

The accumulation of phenolics typically inhibits IAA oxidase, which is consistent with the increased IAA levels often observed in B-deprived plants, which in turn could explain additional symptoms of B deficiency, such as compromised meristematic cell elongation, impaired xylem tissue differentiation, and disrupted lignin biosynthesis. These observations are not always consistent, and the precise relationship between plant B, IAA activity, tissue growth and differentiation, and lignification is not always clear (Marschner 1995), but it is apparent that B nutrition affects these processes.

Boron, in concert with Ca2+, influences reproductive processes, notably the growth of pollen tubes (Mascarenhas and Machlis 1964). Following germination, pollen tubes extend with new tip growth, whereby elongation proceeds by the repeated fusion of vesicles to form new plasma membranes coupled with the continuous secretion of cell wall material. A review of pollen growth suggests that by forming ester complexes with sugar residues on pollen glycoproteins, B essentially guides the synthesis of new membrane and wall material used to sustain pollen tube extension (Blevins and Lukaszewski 1998).

A number of early studies suggested that B plays a causal role in source-to-sink sugar transport. However, these interpretations have been largely discounted, since B only weakly associates with sucrose (Goldbach

1997), the primary sugar transported through the phloem, and the actual phloem-loading physiology is not affected by plant B status. Impaired sugar transport observed in plants with B deficiency likely due to secondary effects that result from declining sink demands when meristematic tissue growth and differentiation are impaired by B deficiency (Marschner 1995). Modified sugar status at growing tips may also reflect the partitioning of carbohydrate metabolism from the glycolytic pathway to the pentose phosphate pathway.

Early literature also offered speculations regarding effects of B nutrition on plant N metabolic processes that ultimately impaired N reduction, leading to reduced NO3 levels and declining amino acid and protein contents in meristematic tissues. However, there is little direct evidence of B involvement in nucleic acid metabolism and protein synthesis (Goldbach 1997).

Marschner (1995) again suggested that altered N status in new growth likely reflects secondary effects stemming from overall declines in sink demands. Mengel and Kirkby (2001) supported this view but postulated that if B played an essential role in uracil synthesis, then a lack of B could impair the formation of RNA products, which would compromise transcription and translation processes underlying protein synthesis and thus lead to impaired meristematic growth processes.

Molybdenum Molybdenum is taken up as the molybdate anion (MoO42−). Although the translocated form is unknown, Mo readily moves through the xylem and phloem.

Molybdenum has the distinction of being the essential plant nutrient present at the lowest concentrations in plant tissues (Table 2.1). The importance of Mo in plant physiology is related to its ability to undergo valency changes (electron transfer) while functioning as an enzyme cofactor. In this capacity, Mo plays several critically important roles in plant N metabolism.

Although plants readily uptake NO3−, further utilization of this N source requires two sequential reductions (NO3− to NO2− and then NO2− to NH3) to produce NH3, which is ultimately assimilated into organic compounds. The initial reduction step is catalyzed by nitrate reductase, a homo-dimer enzyme (composed of two identical subunits), with each subunit harboring three electron-transferring prosthetic groups, including flavin, Fe-heme cytochrome complex, and an Mo cofactor (Römheld and Marschner 1991). Two electrons supplied by either NADH or NADP are accepted by the flavin and transferred to the heme and then to Mo, which ultimately mediates the reduction of NO3− to NO2− (Figure 2.1).

The biological fixation of atmospheric N2 by microorganisms requires the enzymatic activity of nitroge-

THE PHYSIOLOGICAL ROLE OF MINERALS

nase. This enzyme complex includes two Fe-containing proteins: the so-called Fe protein, which harbors a four-Fe−four-S reactive cluster, and the MoFe protein, composed of two subunits, each of which harbors an eight-Fe−seven-S cluster and an MoFe cofactor (Römheld and Marschner 1991) (see Iron). Electrons donated by reducing agents, such as ferredoxin or flavodoxin, are accepted by the Fe protein and then by the MoFe metalloprotein, which not only binds the N2 substrate but also completes the transfer of electrons to N2 (Christiansen et al. 2001). Following the passage of six electrons, nitrogenase releases two NH3 products, completing the fixation process. The molybdenum supply and its impact on nitrogenase activity can significantly influence plant growth, nodule formation, and overall plant N status in nodulated plant species.

Following fixation, the NH3 released by nitrogenase diffuses from the bacteroid complex into the cytoplasm of host plant root cells. In many temperate legume species (lupine, pea, clover, and alfalfa), the NH3 is subsequently assimilated via the GOGAT pathway (Figure 2.2) to produce asparagine as the final N product transported to other plant parts (Marschner 1995). Alternatively, within the root nodule cytoplasm of some tropical and subtropical legumes (soybean, common bean, and cowpea), the NH3 is incorporated into N-rich ureide compounds (notably allantoin and allantoic acid), which are then delivered to the xylem for transport. Within the root cytoplasm, the Mo-containing enzyme xanthine dehydrogenase (with two Mo atoms and eight Fe-S clusters) catalyzes the conversion of the purine xanthine into uric acid, a required precursor of ureide formation (Römheld and Marschner 1991). In this capacity, Mo plays an additional role in the N physiology and metabolism of tropical legume species.

Chlorine The soil solution typically maintains appreciable levels of chlorine as chloride (Cl−). This anionic form is readily taken up by plants, and while the Cl− requirement for plant function and survival is relatively low (Table 2.1), Cl− can accumulate in plants at levels rivaling some macronutrients. Chlorine deficiency is extremely rare, given the ubiquitous presence of Cl− in fertilizers, irrigation water, rainfall, and atmospheric fallout from industrial emissions.

In PS II, Cl− is required for the Hill reaction (Figure 2.7), namely, the series of electron transfers from a four-Mn cluster that ultimately photooxidize water to release oxygen (Kelley and Izawa 1978). Although the exact structural model is not fully understood, the functional assembly of the four-Mn complex and associated proteins requires the presence of Cl− (Merchant

25

and Dreyfuss 1998). Chloride is an effective osmoticum and thus plays a major role in plant and cellular water status. Acting as a charge-balancing anion (often with malate), Cl− moves as a counter-ion with K+ to regulate turgor pressure in guard cells and pulvinus organs, which influence, respectively, stomatal aperture and nyctinastic leaf movements (Satter and Galston 1981).

While proton-pumping ATPases in cell plasma and thylakoid membranes require K+, tonoplastc ATPases require Cl− (Mettler et al. 1982; Churchill and Sze 1984). From this perspective, Cl− plays a key role in the regulation of vacuolar osmotic potential, a driving factor in the expansion and extension of cell volume during cell growth.

Silicon According to the classical definition of essentiality (see Introduction), silicon has yet to be proven an essential nutrient for higher plants. Nonetheless, recent reviews (Epstein 1999) and books (Datnoff et al. 2001; Ma and Takahashi 2002) document a wide range of favorable plant growth development and plant health effects conferred by Si nutrition. Epstein (1999) concluded that “the evidence is overwhelming that in the real world of plant life, Si matters.” Efforts to confirm the essentiality of Si are complicated by the fact that, with the exception of oxygen, Si is the most abundant element in the earth’s crust and is present in almost all soil-forming minerals.

Higher plants can absorb appreciable quantities of silica, evidenced by the mineral analysis of 147 angiosperms (62 monocots and 85 dicots) grown in the same soil, in which the Si contents rivaled and occasionally exceeded Ca, Mg, and P contents (Nishimura et al. 1989, quoted in Epstein 1999). One possibility is that Si functions as a micronutrient, yet testing this hypothesis is problematic, because even the most carefully prepared nutrient solution cultures contain some level of Si contamination. Epstein (1999) proposed that, for many plants, Si should be considered a “quasiessential” element, defined as an element that is ubiquitous in plants, a deficiency of which leads to clearly unfavorable effects or abnormalities in plant growth, development, reproduction, or viability.

Silicon is taken up by roots as monosilicic acid (H4SiO4, or Si(OH)4). The physiology of Si uptake is still not clear, but it is likely that both passive and active uptake mechanisms are involved (Raven 2001).

Once in the xylem, uncharged H4SiO4 readily moves with the transpiration stream and is eventually deposited as solid amorphous silica (SiO2 · nH2O) in the cell wall matrix, cell lumen, and extracellular spaces of shoot, leaf, culm, and root tissues and in the inflorescences of grasses (Sangster et al. 2001).

26 CHAPTER 2

Evidence for the direct involvement of Si in plant metabolic processes is lacking. Nonetheless, Si clearly confers structural, physiological, and/or protective properties that are beneficial to plants subjected to abiotic stresses (environmental effects, such as cold, wind, and mineral uptake imbalances) or biotic stresses (pressures from insects and disease vectors) (Ma et al.

2001). The beneficial effects of Si are particularly realized in plants that readily uptake Si (accumulators), although some nonaccumulator species also accrue benefits with Si nutrition.

The deposition of amorphous silica and formation of opal phytolith structures in epidermal cells and their cell wall matrix confer increased mechanical strength on these tissues. Benefits frequently reported include increased resistance to lodging, particularly in rice and other cereal crops; improved canopy light interception architecture conferred by stiffer shoots and culms and favorably erect leaves; greater tolerance to shearing and compression forces, which could aid root penetration; and improved seed retention in Si-rich bracts (Sangster et al. 2001).

Interactions between Si and Al may lead to the formation of low-solubility aluminosilicates and inert hydroxyaluminosilicates in the cell walls of roots, which effectively reduce levels of toxic Al3+ in the plant (Cocker et al. 1998). Si interacts to create favorable distributions of Mn in leaf tissues, reducing localized accumulations of Mn at toxic levels that would otherwise encourage increased peroxidase activity and tissue necrosis (Jarvis and Jones 1987; Horiguchi and Morita 1987).

In salt-stressed plants, the addition of Si reduced electrolyte losses from plant cells, suggesting that Si may confer a measure of tolerance to salt toxicity (Liang et al. 996). The growth of rice plants cultured in saline environments was improved with Si, a response associated with a sharp reduction in Na translocation from roots to shoots (Matoh et al. 1986). A recent review (Savant et al. 1997) cited numerous studies supporting the view that improved salt stress tolerance in rice is linked to elevated plant Si contents, which in turn favorably influences plant water balances by reducing transpiration (water loss) rates.

Agronomic traits of a number of major world crops, such as rice and sugarcane, are often markedly improved with Si amendments. Indeed, declining yield trends in both plant and ratoon crops have been linked to depletion of natural soil Si reserves (Savant et al. 1997). Sugarcane is widely recognized as a Si accumulator; that is, it readily uptakes Si from the soil solution and clearly displays symptoms of deficiency when soil Si is inadequate. Elawad et al. (1982) reported significant agronomic growth improvements (longer stalks, thicker barrels, increased stalk populations, and improved biomass and sugar yields) in sugarcane

receiving Si amendments, a conclusion independently reached by many investigators worldwide. In a review article, Savant et al. (1997) documented a long list of Si-related agronomic benefits reported for both upland and flooded rice, including healthier or stronger rice seedlings, increased tiller and panicle initiation, a greater number of spikelets, improved grain ripening and grain quality, and increased grain yields. Although the accrual of favorable agronomic traits with Si fertilization fails to highlight any specific physiological function Si may play in plant growth or metabolism, the benefits of Si nutrition are significant and cannot be ignored.

Deposition of Si in cell walls confers mechanical strength and provides a physical barrier against the penetration of fungal plant pathogens and insects.

This argument is widely accepted since the primary sites of Si deposition, namely, cell walls of leaves and the xylem, are also the primary target areas for fungal infection. Additions of Si attenuated the severity of ringspot (caused by Leptosphaeria sacchari) in sugarcane (Raid et al. 1992) and significantly reduced damage by the sugarcane stalk borer (Eldana saccharina), particularly in susceptible varieties (Meyer and Keeping 2001). The severity of fungal diseases in rice, such as rice blast (caused by Magnaporthe grisea), brown spot (caused by Cochliobolus miyabeanus), leaf scald (caused

by Monographella albescens), and sheath blight (caused

by Thanatephorus cucumeris), is reduced with increasing tissue concentrations of Si (Datnoff et al. 1990, 1991; Savant et al. 1997; Seebold et al. 2000). In many cases, Si applications confer levels of disease control equivalent to that obtained by standard fungicide treatments (Datnoff et al. 2001).

While the subcuticular accumulation of Si surely helps protect plants from fungal diseases (and insects), investigators are increasingly considering additional mechanisms of resistance, including the mobilization of host plant defense systems in response to Si. In cucumber, an increasing concentration of Si in leaves was associated with reduced colonization by the powdery mildew fungus Sphaerotheca fuliginea, reduced germination of conidia, and improved plant growth and survival (Menzies et al. 1991a). Although these observations neatly fit into the Si-as-a-barrier theory, follow-up studies suggested that the continued supply of fresh nondeposited Si in the transpiration stream may activate host plant defense mechanisms against the pathogen (Menzies et al. 1991b). This hypothesis was supported by Samuels et al. (1991a), who recorded accumulations of Si around conidia of the pathogen at sites of attempted penetration; the accumulation was associated with new uptake of Si rather than polymerized Si deposits already present in the leaf (Samuels et al. 1991b). In studies of cucumber infected with Pythium ultimum, Si nutrition acted systemically across different plant organs, encouraging the accumulation

THE PHYSIOLOGICAL ROLE OF MINERALS

of phenolic-like compounds that display antifungal properties in infected cells (Chérif et al. 1992b), yet infected sites lacked evidence of Si deposition (Chérif et al. 1992a). These findings raise the possibility that soluble Si may act to induce favorable defense mechanisms in host plants. Addressing these findings in a review article, Fawe et al. (2001) hypothesized that Si may initiate events in cucumber that lead to systemic acquired resistance. Samuels et al. (1991b) reported a rapid loss of induced resistance mechanisms when the Si supply was curtailed. Thus, sustained systemic acquired resistance in cucumber may rely on a relatively constant presence of solubilized Si, which would require continued uptake from the soil solution in order to counteract “losses” due to the deposition of Si in inert forms in epidermal tissues (Fawe et al. 2001).

At present, the essentiality of Si remains unresolved within the scientific community. However, reports documenting a wide range of beneficial aspects of Si nutrition continue to grow in number. At this juncture, a statement by Epstein (1999) is in order: “Taken together, the evidence is overwhelming that silicon should be included among the elements having a major bearing on plant life.”

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