Physiological functions of mineral micronutrients (Cu, Zn, Mn, Fe, Ni, Mo, B, Cl)

Physiological functions of mineral micronutrients (Cu, Zn, Mn, Fe, Ni, Mo, B, Cl)

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Physiological functions of mineral micronutrients (Cu, Zn, Mn, Fe, Ni, Mo, B, Cl) Robert Hansch and Ralf R Mendel

Micronutrients are involved in all metabolic and cellular functions. Plants differ in their need for micronutrients, and we will focus here only on those elements that are generally accepted as essential for all higher plants: boron (B), chloride (Cl), copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo), nickel (Ni), and zinc (Zn). Several of these elements are redoxactive that makes them essential as catalytically active cofactors in enzymes, others have enzyme-activating functions, and yet others fulfill a structural role in stabilizing proteins. In this review, we focus on the major functions of mineral micronutrients, mostly in cases where they were shown as constituents of proteins, making a selection and highlighting some functions in more detail.

Address Institut fur Pflanzenbiologie, Technische Universitat Braunschweig, Humboldtstraße 1, 38106 Braunschweig, Germany

Corresponding author: Mendel, Ralf R (r.mendel@tu-bs.de)

Current Opinion in Plant Biology 2009, 12:259–266

This review comes from a themed issue on Physiology and metabolism Edited by David Salt and Lorraine Williams

1369-5266/$ – see front matter # 2009 Elsevier Ltd. All rights reserved.

Introduction Micronutrients are elements that are essential for plant growthbutarerequiredinmuchsmalleramounts(Table1) than those of the primary nutrients such as nitrogen, phosphorus, sulfur, and potassium. Plants show different needs for certain micronutrients, but the elements that are generally accepted as essential for all higher plants are: boron(B),chloride(Cl),copper(Cu),iron(Fe),manganese (Mn), molybdenum (Mo), nickel (Ni), and zinc (Zn). This list may grow as more protein structures are elucidated. All organisms have to acquire appropriate amounts of each micronutrient that requires a metal homeostasis network involving mobilization, uptake and distribution within the plant, intracellular trafficking, and storage. Several essential metal ions are redox-active that is the basis for their occurrence as catalytically active cofactors in many metalloenzymes. Other metals (like zinc) fulfill in addition to their catalytic role a structural role in stabilizing proteins.

However, when present in elevated concentrations, the same redox properties that make metal ions essential elements lead to the formation of reactive oxygen species with detrimental consequences for the cell. Moreover, metal excess can lead to ectopic binding of metals to proteins thus disturbing protein structures [1]. This beneficial versus detrimental duality caused the development of precisely tuned homeostatic cellular networks involving metal chaperones. One can expect that most metal ions do not exist as free ion in the cell [2 ]. Micronutrientsare involvedinvirtuallyallmetabolicandcellular functions, like energy metabolism, primary and secondary metabolism, cell protection, gene regulation, hormone perception, signal transduction, and reproduction among others. Historically, their physiological role was first described on the basis of deficiency symptoms. In this review, we focus on the major functions of mineral micronutrients, concentrating on cases where the micronutrient is a constituent of a particular protein. The list of proteins given in Table 1 is for some microelements only a short selection (e.g. zinc where we show eight proteins out of over one thousand), for others the list is nearly comprehensive (e.g. molybdenum) and again for others vague and elusive(e.g.boron).Thereaderwillfindmanymoredetails in Marschner’s handbook [3 ] that still is a basic reference for plant nutrients.

Boron The unusual nature of boron chemistry suggests the possibility of a wide variety of biological functions for the micronutrient; however, the exact metabolic functions are not finally understood. Boron is involved in numerous important processes, including protein synthesis, transport of sugars, respiration, RNA and carbohydrate metabolism, and the metabolism of plant hormones (indole acetic acid). Moreover, functions of boron are related to cell wall synthesis, lignification, and cell wall structure by cross-linking of cell wall polysaccharides as well as the structural integrity of biomembranes. It increases the transport of chlorine and phosphorus as a result of plasmalemma ATPase induction. Other investigations have shown that boron can stimulate proton pumping that causes hyperpolarization of the membrane potential (for review see [4]). More than 90% of the boron in plants is found in cell walls, and rhamnogalacturonan I was shown to bind boron [3 ]. Because the wall-associated kinase in the plasmamembrane has an extracellular matrix connection with the pectin molecule [5], the membrane cell wall connection is finally also boron-dependent. Boron was found to w.sciencedirect.com Current Opinion in Plant Biology 2009, 12:259–266

promote the structural integrity of biomembranes and the formation of lipid rafts [3 ]. Since all these functions are fundamental to meristematic tissues, boron deficiency is predominantly damaging actively growing organs such as shoot and root tips so that the whole plant may be stunted (rosetting). Flower retention, pollen formation, pollen tube growth or germination, nitrogen fixation, and nitrate assimilation are also affected by boron [6].

260 Physiology and metabolism

Micronutrients in plants.

Element Symbol Absorbed by plant

Concentration in plant [mgg 1 dry weight]a

Protein complexed with the micronutrient (or other effects)

Literature

Copper (Cu) Cu2+ 1–20 Ascorbate oxidase [49]

Fe–S-cluster Aconitase [5]

Manganese (Mn) Mn2+ 10–100 Mn-superoxide dismutase [51] a The concentration of micronutrients in plants can vary widely depending on the species, genotype, organ, tissue, and growth condition. Therefore ranges are given. b Requirement for optimal growth: 200–400 mgg 1 dry weight.

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Chlorine Chlorine is known to exist in more than 130 organic compounds in plants [7]. Most soils contain sufficient levels of chlorine. However, chlorine deficiencies have been described in sandy soils in high rainfall areas or could be created artificially in experiments to prove its requirement as a micronutrient for higher plants [8]. Because chloride is a mobile anion in plants, most of its functions are related to electrical charge balance. In the chloroplast, chloride is a structural constituent of photosystem I in the oxygen evolving complex as one of the three important cofactors [9]. Proton-pumping ATPase at the tonoplast is specifically stimulated by chloride [10]. The overall chlorine concentration in the whole plant is too low to be an effective osmo-regulator; however, chlorine is accumulated in certain tissues or single cells (e.g. guard cells). Opening and closing of the guard cells is mediated by the flux of potassium and anions such as malate and chloride. Therefore, chlorine indirectly affects plant growth by stomatal regulation. Reduction of leaf surface area, wilting of the plant, and restricted, highly branched root systems are the main chlorine-deficiency symptoms. On the contrary, seismonastic leaf movement of Mimosa pudica is directly chlorine-dependent. The ‘osmotic motor’ for the leaf movement is powered by a plasma membrane proton ATPase, which drives KCl and water fluxes. The movement results from different volume and turgor changes in the two oppositely positioned parts in the specialized motor leaf organs called pulvinus (for details see [1]).

Copper Copper is of utmost importance for life. Copper is essential for photosynthesis and mitochondrial respiration, for carbon and nitrogen metabolism, for oxidative stress protection, and is required for cell wall synthesis, to name only a few of its cellular tasks. Under physiological conditions, copper exists in the two oxidation states Cu1+ and Cu2+ and can interchange between these forms (monovalent copper is unstable). This allows copper to function as a reducing or oxidizing agent in biochemical reactions. But at the same time, this property makes copper also potentially toxic as copper ions can catalyze the production of free radicals, in particular through Fenton chemistry, thus leading to the damage of proteins, DNA, and other biomolecules. Therefore, immediately after uptake the vast majority of copper ions is bound by scavenging proteins like metallothioneins to prevent copper from accumulating in a toxic form. However, part of the imported copper bypasses this system and becomes captured by small binding proteins, so called copper chaperones [2 ,12] that spare copper from the detoxification systems and guide it to the target sites in the cell. If the target is a cytosolic protein, the copper chaperone also directly inserts the metal in the cognate site of the target protein. While in yeast and mammals only a handful of copper chaperones are known, the genome of Arabidopsis thaliana possesses over 30 sequence homologs that might encode for copper chaperones; however, only very few of these proteins are already characterized [13].

In total, more than 100 Arabidopsis proteins are predicted to be complexed with copper [14 ], comprising two groups: copper binding proteins/chaperones and enzymes. Copper has a particularly high affinity to dioxygen molecules that explains why copper is the catalytic metal in many oxidases. The most prominent member of this group is mitochondrial cytochrome c oxidase as principal catalyst of the terminal oxidation. Copper is also found in electron carrier proteins like plastocyanin that accounts for about 50% of the plastidic copper [15]. More than half of the copper in plants is found in chloroplasts and participates in photosynthetic reactions. Hence, copper deficiency becomes first visible in young leaves and reproductive organs, later consequences are stunted growth of the whole plant and pale green leaves that wither easily. Interestingly, copper metabolism is intimately linked to iron metabolism. Depending on the bioavailability of copper and iron, plants possess enzymes for the alternative use of copper versus iron thus catalyzing the same biochemical reaction with completely different apoproteins [16], a process that involves regulation by miRNAs [17 ]. Examples include Cu-nitrite versus heme-nitrite reductase, Cu/Zn-superoxide dismutase versus Fe-superoxide dismutase, and cytochrome oxidase versus diiron oxidase. Finally, copper is also part of the ethylene receptor [18] and is involved in molybdenum cofactor biosynthesis [19].

Iron Like copper, iron is also of great importance for life. As redox-active metal it is involved in photosynthesis, mitochondrial respiration, nitrogen assimilation, hormone biosynthesis (ethylene, gibberellic acid, jasmonic acid), production and scavenging of reactive oxygen species, osmoprotection, and pathogen defense. Up to 80% of the cellular iron is found in the chloroplasts that is consistent with its major function in photosynthesis. Depending on the type of iron ligand, three groups of iron-containing proteins can be defined: (1) proteins with iron–sulfur clusters (Fe–S), (2) heme-containing proteins, and (3) other iron proteins.

Fe–S proteins:F e–S clusters have pivotal functions in electron transfer, they constitute part of substrate binding sites in enzymes, they form iron storage moieties, they are involved in transcriptional or translational regulation, they can control protein structure in the vicinity of the cluster, and finally they have been shown to be involved in disulfide reduction and sulfur donation (e.g. thioredoxins). Hence, Fe–S proteins serve functions as enzymes, as electron carriers (e.g. ferredoxin), and as regulator proteins (e.g. aconitase). Fe–S cluster formation occurred very early in evolution and was strictly conserved later on.

Physiological functions of mineral micronutrients (Cu, Zn, Mn, Fe, Ni, Mo, B, Cl) Hansch and Mendel 261 w.sciencedirect.com Current Opinion in Plant Biology 2009, 12:259–266

Fe–S clusters are built from inorganic iron and sulfide. While in vitro they can form spontaneously under anaerobic conditions, the low concentrations of both components in the cell preclude a spontaneous assembly. Therefore, a complex assembly system has been evolved in bacteria that have counterparts in mitochondria and chloroplasts [20]. The cytoplasm receives its Fe–S clusters from the mitochondria.

Heme proteins: The well-known hemoproteins are the photosynthetic and respiratory cytochromes, involved in electron transfer, and the globins that bind oxygen. Other examples include the oxidative enzymes catalase, peroxidase, and NADPH oxidase, involved in the production and/or scavenging of free radicals, and the very large group of cytochrome P450 enzymes. In plants and microbes, these latter catalyze mono-oxygenation reactions in biosynthetic pathways, such as for sterols and many secondary metabolites, whereas in animals their major role is in the detoxification of xenobiotics. Further, globins like leg-hemoglobin are involved in oxygen binding and transport. Nitrite reductase and sulfite reductase harbor both a siroheme and an Fe–S cluster in the enzyme. Little is known about the coordination between apoprotein and heme synthesis or the assembly into a functional protein. In plants, hemoproteins are distributed in all subcellular locations, but the situation is made more complex by the fact that heme is synthesized in both mitochondria and chloroplasts [21], and it is not known which organelle supplies heme to other users in the cell. For example, cytochrome P450 localizes to the endoplasmic reticulum, catalase to the peroxisomes and other enzymes to the cytoplasm.

Other iron proteins: These proteins (that are sometime also grouped as non-heme proteins) bind iron ions directly, i.e. neither as heme nor in the Fe–S form. Among these proteins, ferritins are most prominent. Ferritins are plastidic iron storage proteins and control the interaction between iron homeostasis and oxidative stress in Arabidopsis [2]. They are high molecular weight 24-mer proteins that can store up to 4500 ion atoms in a soluble and bioavailable form [23]. Ferritins occur mostly in nongreen plastids like etioplasts and amyloplasts but not in mature chloroplasts [24].

Manganese Manganese is essential for plant metabolism and development and occurs in oxidation states I, II, and IV in approximately 35 enzymes of a plant cell [25]. Manganese can fulfill two functions in proteins: (1) it serves as catalytically active metal, or (2) it exerts an activating role on enzymes. Examples for the catalytic role are manganese-containing superoxide dismutase protecting the cell from damaging effects of free radicals, the oxalate oxidase, and the manganese-containing water splitting system of photosystem I [26]. Examples for the manga- nese-activated enzymes are malic enzyme, isocitrate dehydrogenase, PEP carboxykinase, and phenylalanin ammonia lyase. Among the rather large group of manganese-activated enzymes, the role of manganese is less specific as in many cases it can be replaced by magnesium [3 ]. Proteins belonging to this group are involved in the shikimic acid pathway and subsequent pathways leading to the formation of aromatic amino acids, lignins, flavonoids, and the phytohormone indole acetic acid. Manganese activation was seen in enzymes of nitrogen metabolism (glutamin synthetase, arginase), gibberellic acid biosynthesis, RNA polymerase activation, and fatty acid biosynthesis.

Molybdenum Only a handful of plant proteins are known to contain molybdenum. These proteins, however, are very important as they are involved in nitrogen assimilation, sulfur metabolism, phytohormone biosynthesis, and stress reactions [27]. Nitrate reductase is the key-enzyme for nitrate assimilation while nitrogenase is found in nitrogen fixing bacteria inside nodules of symbiotically growing species. The last step of abscisic acid biosynthesis is catalyzed by the molybdenum-enzyme aldehyde oxidase, and sulfite oxidase protects the plant against toxic levels of sulfite (acid rain!). Hence a defect in molybdenum-metbolism leads to the pleiotropic loss of these enzyme activities with lethal consequences for the organism. Recently, a novel molybdenum-enzyme (‘mitochondrial amidoxime reducing component’ mARC) was found on the envelope of mammalian mitochondria [28] catalyzing the reduction of N-hydroxylated amidines in concert with cytochrome b5 and cytochrome b5 reductase, a reaction that may be associated with detoxification tasks. This new enzyme is wide spread in nature as homologs were found among plants and eubacteria [29]. In all organisms, molybdenum has to be complexed by a pterin compound thereby forming the molybdenum cofactor in order to gain biological activity. This pterin compound is a unique pterin named molybdopterin or metal-containing pterin. With the exception of nitrogen-fixing nitrogenase all molybdenum-containing enzymes characterized to this end contain the pterin-type cofactor [30]. Interestingly, molybdenum enzymes produce reactive oxygen species (sulfite oxidase, aldehyde oxidase, xanthine dehydrogenase) or NO (nitrate reductase) as side reaction products. Molybdenum metabolism is intimately linked to iron and copper metabolism at several crosspoints (Figure 1). In all organisms, enzymes participating in the first step of molybdenum cofactor biosynthesis were found to contain Fe–S clusters. Further down the molybdenum pathway, nitrogenase, aldehyde oxidase, and xanthine dehydrogenase bind Fe–S clusters as well. And also nitrate reductase contains iron in the form of the heme group. Another crosstalk was discovered between molybdenum and copper metabolism as copper was found to be essential for the formation of a molybdenum cofactor intermediate [19].

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Nickel Nickel is essential in numerous prokaryotic enzymes like dehydrogenases, hydrogenases, and methylreductases but is barely used as cofactor in eukaryotes. Among plants, it occurs not only in oxidation states I, but also in states I and I. A deficiency symptom in plants is the accumulation of toxic urea [31–3]t hat could be explained with the complete loss of urease activity within the cell. Plant urease hydrolyzes its substrate to carbon dioxide and ammonia. Moreover, recent findings of Follmer suggest that plant ureases have a protective role against phytopathogens, unrelated to the release of ammonia [34]. The bacterial homotrimeric or homotetrameric enzyme contains two Ni2+- ions per subunit in its catalytic center [35]. An additional Ni2+-binding protein could be identified in soybean that acts as Ni-metallochaperone essential for urease activity [36]. It seemst ob ep ossiblet hataf ewm ore

Physiological functions of mineral micronutrients (Cu, Zn, Mn, Fe, Ni, Mo, B, Cl) Hansch and Mendel 263 Figure 1

Schematic representation of the metabolic link between the three micronutrients molybdenum, iron, and copper as it is found in molybdenum metabolism. In order to become biologically active, molybdenum has to be complexed by a pterin compound thereby forming the molybdenum cofactor (Moco). The first step of Moco biosynthesis taking place inside mitochondria is dependent on Fe–S clusters that form part of the enzymes involved in this step. Mitochondria are the site of Fe–S cluster synthesis and support the biogenesis of extra-mitochondrial Fe–S proteins by exporting Fe–S cluster equivalents through a specific transporter into the cytosol. There are indications that this transporter is also involved in exporting a Moco intermediate. Mitochondria synthesize and export heme groups as well. With the exception of sulfite oxidase and mARC, molybdenum-containing enzymes also need iron, either in the form of Fe–S clusters or as heme-iron. Finally, copper is involved in Moco biosynthesis by facilitating molybdenum insertion into the cofactor precursor. In summary, molybdenum metabolism is fully dependent on a functional iron and copper metabolism.

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Ni-dependent enzymes will be identified in plants in the future.

Zinc Zinc is important as a component of enzymes for protein synthesis and energy production and maintains the structural integrity of biomembranes. More than 1200 proteins are predicted to contain, bind, or transport Zn2+, including – among others – a large numbers of zinc-finger containing proteins and transcription factors, oxidoreductases, and hydrolytic enzymes such as metalloproteases [14 ]. Zinc plays also an important role in seed development, and zinc-deficient plants show a delayed maturity.

Most of the zinc enzymes are involved in regulation of DNA-transcription, RNA-processing, and translation. Up to 4% of the predicted proteins in Arabidopsis contain the zinc-finger motif [37] that is well known in transcription factors and is important for protein–protein interactions. Beside transcription factors, several enzymes involved in DNA or RNA synthesis and maintenance are zinc-dependent: all types of DNA-polymerases and RNA-polymerases, histone deacetylases, splicing factors, and enzymes involved in RNA-editing in mitochondria and chloroplasts [14 ]. Furthermore, zinc was found in a number of tRNA synthetases and in the translation initiation factor elF-5 from maize that is required for the formation of a functional 80S initiation complex [38].

In chloroplasts, zinc-dependent enzymes fulfill several major functions. The stromal processing peptidase SPP [39] is zinc-dependent in analogy to the mitochondrial system [40]. Additional zinc metalloproteases in these organelles have to destroy the cleaved signal-peptide [41]. Moreover inside the chloroplasts proteolytic activities are dependent on zinc, for example, the repair processes of photosystem I by turning over photo-damaged D1 protein [42]. Zinc deficiency also reduces net photosynthesis in plants by disturbing the activity of carbonic anhydrase, which is the limiting enzyme for CO2 fixation in C4-plants, Cu–Zn superoxide dismutase, and D-ribu- lose-5-phosphate 3-epimerase, an enzyme of the Calvin cycle and oxidative pentose phosphate pathway [43].

In addition to chloroplasts and mitochondria, also the cytoplasm, lysosome, and the apoplastic space are compartments with zinc-dependent hydrolytic activities: different nucleases and aminopeptidases, peptide deformylases [4], the 26S-proteasome [45], the a-mannosidase [46], and matrix-metalloproteinases associated with the extracellular matrix [47]. Further, Zinc was found to be involved in signal transduction via mitogen-activated protein kinases [48 ].

Conclusion Essential micronutrients were found as constituents in over 1500 proteins where they fulfill catalytic, (co-)acti- vating, and/or structural functions. The largest group (>1200) is formed by zinc-proteins (with transcription factors as major subgroup). Proteins containing iron, copper, or manganese make up groups in the range of 50–150 members each, while molybdenum and nickel proteins can be counted on one hand each. Boron and chlorine are very important, but proteins or compounds that were unambiguously shown to contain these micronutrients are very rare and mostly elusive.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

of special interest of outstanding interest

1. Yang M, Cobine PA, Molik S, Naranuntarat A, Lill R, Winge DR,

Culotta VC: The effects of mitochondrial iron homeostasis on cofactor specificity of superoxide dismutase 2. EMBO J 2006, 25:1775-1783.

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