Plant Nutrition 3: Micronutrients and Metals

Non-metals Essential Metalloids micronutrients Metals D-block metals

Non-essential toxic elements (examples)

Essential for animals, beneficial for

www.plantcell.org/cgi/doi/10.1105/tpc.109.tt1009plants

© 2015 American Society of Plant Biologists Lesson outline • Introduction to micronutrients The hydroponics system developed by Hoagland and Arnon for the characterization of micronutrients • Micronutrient transporters and transport • Essential metal micronutrients • Fe, Zn, Cu, Mn, Mo, Ni • Metal tolerance and metal hyperaccumulation • Toxic metals and metalloids • As, Cd, Al • Other micronutrients • B, Cl, Si, Se • Summary and ongoing research

Dennis Hoagland and colleagues developed a soil- free system for micronutrient studies. Today, “Hoagland’s Solution” continues to be used as a complete plant nutrient solution.

Adapted from Hoagland, D.R., and Arnon, D.I. (1950).The water-culture method for growing plants without soil. Circular. California Agricultural Experiment Station. Volume 347. 2nd edition.

“An element is not considered essential Or… “the plant can be so severely deficient unless a deficiency of it in the element that it exhibits abnormalities makes it impossible for in growth, development, or reproduction, i.e. the plant to complete its "performance," compared to plants with a life cycle” Mn lower deficiency” -Arnon and Stout, 1939 B -Epstein and Bloom, 2003 Zn Cu Fe Mo Cl 1969 Ni 1920 - 1954 1987 1860 Si 1940 (Required for Equisetum)

Year when each element was shown to be essential

Arnon, D.I., and Stout, P.R. (1939). The essentiality of certain elements in minute quantity for plants with special reference to copper. Plant Physiol. 14: 371 – 375; Epstein, E. and Bloom, A.J. (2003). Mineral Nutrition of Plants: Principles and Perspectives, 2nd Ed., John Wiley & Sons, New York. See also Marschner, P., ed (2012). Marschner’s Mineral Nutrition of Higher Plants. 3rd ed. (London: Academic Press).

Micronutrient availability There are exceptions, but most plants grow best at a slightly acid pH where the availability of nutrients is generally high. In strongly acidic soils, growth is often limited by increased solubility of the abundant but detrimental non-nutritive element aluminum

Aluminum

Example: Iron is more soluble in acidic soils

+ 3+ Fe(OH)3 + 3H Fe + 3H2O Strongly Mildly Insoluble Soluble acidic alkaline

Deficiency Luxury (plants (plants can can store or adjust) detoxify)

Limitation Toxicity Tolerance Toxicity

Plant dry weight dry Plant Plant dry weight dry Plant

Concentration of element Concentration of element

Adapted from Lin, Y.-F. and Aarts, M.M. (2012). The molecular mechanism of zinc and cadmium stress response in plants. Cellular and Molecular Life Sciences. 69: 3187-3206 and Merchant, S.S., and Helmann, J.D. (2012). Elemental economy: Microbial strategies for optimizing growth in the face of nutrient limitation. Adv. Microb. Physiol 60: 91 – 210.

Element Biologically Concentration in plant relevant form in (mg / kg) plants (These values vary by species, other nutrient levels etc.) Deficiency Normal Toxicity Iron (Fe) Fe2+, Fe3+ < 20 20 – 1000 > 2000 For comparison, Copper (Cu) Cu+, Cu2+ < 10 10 – 25 > 25 concentrations of Zinc (Zn) Zn2+ < 10 10 – 120 > 120 macronutrients Manganese (Mn) Mn2+, Mn3+, Mn4+ < 90 90 – 200 > 200 range from 1000 –

Molybdenum Mo4+, Mo6+ (in < 0.1 0.1 – 90 > 90 450,000 mg / kg) (Mo) Moco or FeMoco)

Boron B(OH)3 < 10 10 – 80 > 80 Chloride Cl- > 100 100 – 800 > 800

Nickel (Ni) Ni2+ > 0.05 0.05 – 10 > 10

Palmer, C.M. and Guerinot, M.L. (2009). Facing the challenges of Cu, Fe and Zn homeostasis in plants. Nat Chem Biol. 5: 333-340. See also Marschner, P., ed (2012). Marschner’s Mineral Nutrition of Higher Plants. 3rd ed. (London: Academic Press) and Krämer, U. (2010). Metal hyperaccumulation in plants. Annu. Rev. Plant Biol. 61: 517-534.

Limitation Deficiency Luxury Toxicity

Optimal range

Plant dry Plant weight

Concentration of element

Increased Efflux to Alternative uptake: apoplast metabolic (upregulated pathways transporters, Storage secretion of (organelle) Recycling siderophores and chelators) Chelation / sequestration

© 2015 American Society of Plant Biologists Nutrient deficiency characteristics illustrate nutrient function Zinc is required for growth Iron and Mn are both particularly of young needed for tissues. Deficiency chlorophyll causes stunting and the production. formation of small leaves (“little leaf”) Deficiency of either of these poorly mobile elements causes interveinal chlorosis (yellowing)

Copper is required for cell energetics including photosynthesis and respiration. Deficiency symptoms include chlorosis and necrosis of young tissues and leaf tips

Photo credits: William M. Ciesla, Forest Health Management International, Bugwood.org; CropNutrition

Metal micronutrients

Metals D-block metals

Bar height = % of cellular proteins Enzymes that use Fe, Cu or Mo Macro- requiring element as cofactor are mainly oxidoreductases nutrients (blue bars)

Micronutrients

Enzymes that use Mn, or Zn have more diverse functions: These data are obtained from a Yellow= transferases; purple= protein database representing hydrolase; red= lyase; green= isomerase; grey= ligase multiple organisms

Reprinted by permission from Macmillan Publishers Ltd: Waldron, K.J., Rutherford, J.C., Ford, D. and Robinson, N.J. (2009). Metalloproteins and metal sensing. Nature. 460: 823-830.

− Fenton reaction About ¼ of cellular proteins e (n)+ are metalloproteins (not M + H2O2 counting those that make loose associations with Mg) M(n)+ M(n+1)+ M(n+1)+ + HO· + OH−

− HO· (hydroxyl radical) is highly reactive e− Several metals can alternate between being electron donors and acceptors, Uncontrolled metal reactions can damage extending the range of cells for example through reactions that can occur in free radical production The apoprotein is biological systems. the polypeptide part without the metal This particularly applies to Fe(II) and Fe (III) and Cu(I) and Cu(II)

Root vacuole Shoot vacuole Shoot & other Root apoplast & other apoplast pool organelle pools pool organelle pools

Efflux into Uptake (influx) xylem Bioavailable into root apoplast Shoot pool in Root symplast rhizosphere symplast pool Efflux from pool Xylem Influx into root apoplast symplast pool Small molecules Phloem Sink tissues Effects of released from symplast (e.g., seeds) soil root to increase pool microbes nutrient availability Indicates that a transporter is required

Nutrients must be Membrane transport can transported across Cross membrane consume 1/3 of a cell’s into living cell in metabolic energy (or more) membranes to enter root hair the plant Symplastic or transcellular pathway Casparian strip

Bidirectional transport Pumps, channels Apoplastic pathway between xylem parenchyma and carriers are the Cross membrane cells and apoplastic molecular into living cell in transpiration stream mediators of these at endodermis processes

Zn, Cd Mn A nutrient Cu transported solely by transpiration, would accumulate in leaves (sites of most transpiration)

Nutrients need to move into phloem (through transporters) for distribution to young growing tissues

M(n)+ Metal micronutrients are bound by chelators (small molecules or proteins) during transport

Reprinted from Yamaji, N. and Ma, J.F. (2014). The node, a hub for mineral nutrient distribution in graminaceous plants. Trends Plant Sci. 19: 556-563 with permission from Elsevier.

Apoplast to cytosol to vacuole and vice versa

Soil to root Into and out of apoplastic xylem

Reprinted from Clemens, S., Palmgren, M.G. and Krämer, U. (2002). A long way ahead: understanding and engineering plant metal accumulation. Trends Plant Sci. 7: 309-315 with permission from Elsevier.

Most metal NRAMPs. Metal influx transporters can into cytosol transport multiple metals, so their activity depends in part on nutrient HMAs: Heavy Metal concentration Transporting ATPases. Pump metals outwards YSLs: Yellow from cytosol Stripe1- Like. Metal-chelate influx into cytosol

MTPs: Metal Tolerance ZIPs: Zinc, iron related Proteins. Metal efflux transporter. Metal influx from cytosol into cytosol Reprinted from Krämer, U., Talke, I.N. and Hanikenne, M. (2007). Transition metal transport. FEBS letters. 581: 2263-2272.

George Beadle (foreground) with other The yellow-stripe1 mutant Along with Edward Tatum, maize geneticists, 1929 Ithaca New York (right) has yellow stripes Beadle was awarded the between the veins due to a Nobel Prize in Physiology lack of chlorophyll or Medicine (1958)…

“for their discovery that genes act by regulating yellow definite chemical events.” stripe Beadle and Tatum used the In the 1950s and 1960s, fungus Neurospora to show Lawrence Bogorad’s group that relationship between showed that this phenotype genes and proteins. Although Beadle went on to is caused by iron deficiency. work on many genetic systems, The transporter was cloned his roots are in maize genetics in 2001

See Beadle, G.W. (1929). Yellow-Stripe-a Factor for chlorophyll deficiency in maize located in the Pr pr chromosome. Am. Nat. 63: 189 – 192. Reprinted by permission from Macmillan Publishers Ltd: Curie, C., Panaviene, Z., Loulergue, C., Dellaporta, S.L., Briat, J.-F., and Walker, E.L. (2001). Maize yellow stripe1 encodes a membrane protein directly involved in Fe(III) uptake. Nature. 409: 346-349. Bell, W.D., Bogorad, L., McIlrath, W.J. (1958). Response of the yellow-stripe mutant (ys1) to ferrous and ferric iron. Bot. Gazz. 120: 36 – 39 Beadle

© 2015 American Society of Plant Biologists Group B transporters move Some micronutrients transporters out of the (Group A) cytosol (or from move the inter- micronutrients membrane space into the into the stroma cytosol or matrix)

Reprinted from Blaby-Haas, C.E. and Merchant, S.S. (2012). The ins and outs of algal metal transport. Biochim. Biophys. Acta. 1823: 1531-1552.

© 2015 American Society of Plant Biologists Some transporters move nutrients into and out of organelles Proteins and processes are color coded: iron (Fe), copper (Cu), zinc (Zn), manganese (Mn), cobalt (Co) and molybdenum (Mo)

Vacuole

Candidate transporters

Nouet, C., Motte, P. and Hanikenne, M. (2011). Chloroplastic and mitochondrial metal homeostasis. Trends Plant Sci. 16: 395-404; See also Puig, S. and Peñarrubia, L. (2009). Placing metal micronutrients in context: transport and distribution in plants. Curr. Opin. Plant Biol. 12: 299-306 with permission from Elsevier.

© 2015 American Society of Plant Biologists Chelators & chaperones bind metals so they don’t react inappropriately Chelators grasp metal ions like a crab’s claw. “Chela” in Greek refers to a grasping organ like a crab’s claw

Ferrous malate Chaperones are small metal-binding proteins

For example + 3+ Fe(OH)3 + 3H Fe + 3H2O Soluble Insoluble citrate Iron citrate

Reproduced from Banci, L., Bertini, I., McGreevy, K.S. and Rosato, A. (2010). Molecular recognition in copper trafficking. Natural Prod. Rep. 27: 695-710

Siderophores are structurally diverse. 1. Bacteria and They are defined other organisms functionally as a small secrete molecule that binds siderophores when A siderophore, metals (the term iron is limiting enterobactin literally means “iron carrier”) Fe3+ + 2. The siderophore binds (chelates) the metal, keeping the metal in solution Uptake 3. The siderophore- metal complex Fe-enterobactin imported into the cell

Reproduced from Blindauer, C.A. and Schmid, R. (2010). Cytosolic metal handling in plants: determinants for zinc specificity in metal transporters and metallothioneins. Metallomics. 2: 510-529 with permission of The Royal Society of Chemistry.

COOH COOH COOH COOH 3 x The enzyme In grasses, phytosiderophores nicotiananamine are produced from NA, and NH + NH N NH2 S 2 synthase (NAS) released from the plant into the Nicotianamine Adenosyl makes soil to enhance metal uptake nicotianamine (NA) from three Metal-PS molecules of S- complex COOH COOH COOH COOH COOH COOH Yellow= metal adenosyl Red = oxygen N NH OH methionine Blue = nitrogen NH N NH2 Green = carbon Phytosiderophore Nicotianamine Grasses have a suite of enzymes that converts NA to various compounds in the PS family NA chelates Citrate and other small molecules are also metal chelators metals for HN

transport within N O

the plant Citrate O − Metal-NA N Ni H2 complex + N

− O

H2 O

Purple = metal Metal N

Red = oxygen Ni(His)

Blue = nitrogen Fe(III)-citrate HN

Blindauer, C.A. and Schmid, R. (2010). Cytosolic metal handling in plants: determinants for zinc specificity in metal transporters and metallothioneins. Metallomics. 2: 510-529.

Phytochelatins Metallothioniens

Phytochelatins (PCs) are sulfur-rich peptides synthesized enzymatically from glutathione. Their primary role is to confer protection from excess metals by binding them in the cytosol or sequestering them in the vacuole

Phytochelatin Yellow= sulfur Red = oxygen Blue = nitrogen Grey = carbon White = hydrogen Metallothionines are Cys-rich small proteins encoded by genes. Some have metal storage functions, others may be involved in detoxification

Blindauer, C.A. and Schmid, R. (2010). Cytosolic metal handling in plants: determinants for zinc specificity in metal transporters and metallothioneins. Metallomics. 2: 510-529.Rea, P.A., Vatamaniuk, O.K. and Rigden, D.J. (2004). Weeds, worms, and more. Papain's long-lost cousin, phytochelatin synthase. Plant Physiol 136: 2463-2474; Leszczyszyn, O.I., Imam, H.T. and Blindauer, C.A. (2013). Diversity and distribution of plant metallothioneins: a review of structure, properties and functions. Metallomics. 5: 1146-1169..

Iron is the fourth most Oxygenic photosynthesis, beginning ~ 2.5 billion years abundant element in the earth’s crust ago, rendered Fe largely oxidized and insoluble. For most cells, obtaining sufficient Fe is a challenge Rest Na Mg Fe K 3+ Ca Fe(II) or Fe2+ Oxygen Fe(III) or Fe Ferrous ion Ferric ion Soluble Insoluble Al O Si

Reprinted by permission from Macmillan Publishers Ltd Rasmussen, B., Fletcher, I.R., Bekker, A., Muhling, J.R., Gregory, C.J. and Thorne, A.M. (2012). Deposition of 1.88-billion-year-old iron formations as a consequence of rapid crustal growth. Nature. 484: 498-501.

Iron doesn’t move well in the plant, so is more concentrated around the veins. Iron deficiency interferes with chlorophyll production, resulting in interveinal chlorosis

Iron-requiring enzyme

Chlorophyll

MgP monomethyl Divinyl ester (MgPMME) protochlorophyllide (Pchlide)

Tottey, S., Block, M.A., Allen, M., Westergren, T., Albrieux, C., Scheller, H.V., Merchant, S. and Jensen, P.E. (2003). Arabidopsis CHL27, located in both envelope and thylakoid membranes, is required for the synthesis of protochlorophyllide. Proc. Natl. Acad. Sci. USA. 100: 16119-16124; William M. Ciesla, Forest Health Management International, Bugwood.org.

Or flavodoxin (Cu)

Respiratory electron transport in Or plastocyanin (Cu) mitochondria

Photosynthetic electron transport in chloroplasts

Reprinted from Blaby-Haas, C.E. and Merchant, S.S. (2013). Iron sparing and recycling in a compartmentalized cell. Curr. Opin. Microbiol. 16: 677-685 by permission from Elsevier

© 2015 American Society of Plant Biologists Iron in cells is found in heme, Fe-S clusters and other forms In cells, iron is found in many forms, including heme, siroheme, Fe-S clusters (mainly Fe2S2 and Fe4S4), di-iron centers, mononuclear Fe and others (e.g., sulfite reductase, Ferrodoxin-nitrite reductase)

The hormone FeSOD, found in plastids, also has a ethylene (C2H4) is synthesized by the mononuclear nonheme Fe (red enzyme ACC oxidase sphere) at the which uses a reaction center mononuclear (This structure is nonheme Fe(II) center from Plasmodium)

Reprinted from Balk, J. and Schaedler, T.A. (2014). Iron cofactor assembly in plants. Annu. Rev. Plant Biol. 65: 125-153 with permission; Schofield, C.J. and Zhang, Z. (1999). Structural and mechanistic studies on 2- oxoglutarate-dependent oxygenases and related enzymes. Curr. Opin. Struct. Biol. 9: 722-731; Rocklin, A.M., Tierney, D.L., Kofman, V., Brunhuber, N.M.W., Hoffman, B.M., Christoffersen, R.E., Reich, N.O., Lipscomb, J.D. and Que, L. (1999). Role of the nonheme Fe(II) center in the biosynthesis of the plant hormone ethylene. Proc. Natl. Acad. Sci. USA 96: 7905-7909; Boucher, I., Brzozowski, A., Brannigan, J., Schnick, C., Smith, D., Kyes, S. and Wilkinson, A. (2006). The crystal structure of superoxide dismutase from Plasmodium falciparum. BMC Struct. Biol. 6: 20.

Fe solubilization and chelation

Iron deficiency Reduction of induces proton Fe(III) to Fe (II) extrusion and the export of phenolics such as scopoletin Fe import

Reprinted from Brumbarova, T., Bauer, P. and Ivanov, R. (2015). Molecular mechanisms governing Arabidopsis iron uptake. Trends Plant Sci. 20: 124-133 with permission from Elsevier; Fourcroy, P., et al., and Briat, J.-F. (2014). Involvement of the ABCG37 transporter in secretion of scopoletin and derivatives by Arabidopsis roots in response to iron deficiency. New Phytol. 201: 155-167.

This strategy is restricted to grasses because only grasses convert nicotianamine (NA) to Fe(III) chelated to phytosiderophore 2′-deoxymugineic acid (DMA) and derivatives (also known as phytosiderophores)

Reprinted from Kobayashi, T., Nakanishi Itai, R. and Nishizawa, N. (2014). Iron deficiency responses in rice roots. Rice. 7: 27. See also Kobayashi, T. and Nishizawa, N.K. (2012). Iron uptake, translocation, and regulation in higher plants. Annu. Rev. Plant Biol. 63: 131-152; Fourcroy, P., Sisó-Terraza, P., Sudre, D., Savirón, M., Reyt, G., Gaymard, F., Abadía, A., Abadia, J., Álvarez-Fernández, A. and Briat, J.-F. (2014). Involvement of the ABCG37 transporter in secretion of scopoletin and derivatives by Arabidopsis roots in response to iron deficiency. New Phytol. 201: 155-167.

OPT3 is a phloem localized transporter that moves Fe into phloem, linking xylem and phloem transport, and providing roots with information about shoot Fe status

Zhai, Z., Gayomba, S.R., Jung, H.-i., Vimalakumari, N.K., Piñeros, M., Craft, E., Rutzke, M.A., Danku, J., Lahner, B., Punshon, T., Guerinot, M.L., Salt, D.E., Kochian, L.V. and Vatamaniuk, O.K. (2014). OPT3 is a phloem-specific iron transporter that is essential for systemic iron signaling and redistribution of iron and cadmium in Arabidopsis. Plant Cell. 26: 2249-2264.

FRD3 is a citrate efflux transporter Citrate keeps the metal soluble in the apoplast; Variation in moving from root cells expression into xylem apoplast, levels of FRD3 moving from xylem affect Fe apoplast into leaf cells, homeostasis and moving iron into and Zn seeds and pollen tolerance

Roschzttardtz, H., Séguéla-Arnaud, M., Briat, J.-F., Vert, G. and Curie, C. (2011). The FRD3 citrate effluxer promotes iron nutrition between symplastically disconnected tissues throughout Arabidopsis development. Plant Cell. 23: 2725-2737. See also Charlier, J.-B., Polese, C., Nouet, C., Carnol, M., Bosman, B., Krämer, U., Motte, P. and Hanikenne, M. (2015). Zinc triggers a complex transcriptional and post-transcriptional regulation of the metal homeostasis gene FRD3 in Arabidopsis relatives. J. Exp. Bot. doi: 10.1093/jxb/erv188. Pineau, C., Loubet, S., Lefoulon, C., Chalies, C., Fizames, C., Lacombe, B., Ferrand, M., Loudet, O., Berthomieu, P. and Richard, O. (2012). Natural variation at the FRD3 MATE transporter locus reveals cross-talk between Fe homeostasis and Zn tolerance in Arabidopsis thaliana. PLoS Genet. 8: e1003120.

Fe2+ is oxidized to insoluble Fe3+ for storage

Ferritin forms a protein shell that stores and sequesters iron

Plastid-localized ferritins can store up to 80% of total

leaf iron, providing a significant Fe sink and buffer [FeO(OH)]8[FeO(H2PO4)]

Reprinted from Lewin, A., Moore, G.R. and Le Brun, N.E. (2005). Formation of protein-coated iron minerals. Dalton Transactions. 3597-3610 with permission from Royal Society of Chemistry See also Briat, J.-F., Duc, C., Ravet, K. and Gaymard, F. (2010). Ferritins and iron storage in plants. Biochimica et Biophysica Acta (BBA) - General Subjects. 1800: 806-814.; WUSTL.

The transcription factor FIT co-ordinately regulates several Fe-uptake genes

Additional transcription factors and other regulators contribute to iron homeostasis

AHA2 = H+ pump acidifies rhizosphere FRO2 = Ferric reductase IRT1 = Iron uptake transporter

Reprinted from Brumbarova, T., Bauer, P. and Ivanov, R. (2015). Molecular mechanisms governing Arabidopsis iron uptake. Trends Plant Sci. 20: 124-133 with permission from Elsevier.

The plant’s iron status affects the circadian period, and many iron-uptake genes are under circadian control.

Given that iron proteins are required for photosynthesis and also detoxification of reactive oxygen, these controls may help coordinate and optimize metabolic processes.

Reprinted from Salomé, P.A., Oliva, M., Weigel, D. and Krämer, U. (2013). Circadian clock adjustment to plant iron status depends on chloroplast and phytochrome function. The EMBO Journal. 32: 511-523; Hong, S., Kim, S.A., Guerinot, M.L. and McClung, C.R. (2013). Reciprocal interaction of the circadian clock with the iron homeostasis network in Arabidopsis. Plant Physiology. 161: 893-903; Chen, Y.-Y., Wang, Y., Shin, L.-J., Wu, J.-F., Shanmugam, V., Tsednee, M., Lo, J.-C., Chen, C.-C., Wu, S.-H. and Yeh, K.-C. (2013). Iron is involved in the maintenance of circadian period length in Arabidopsis. Plant Physiol. 161: 1409-1420.

Cytosol Fe deficit It’s not clear Iron deficiency how iron is induces iron uptake sensed in genes and metal plants Vacuole FIT homeostasis genes Iron deficiency Iron uptake genes triggers expression Metal-homeostasis of vacuolar metal genes IRT1 can be internalized sequestration from the plasma membrane or subjected to genes FRO2 MTP proteolytic degradation to IRT1 prevent uptake of excess FeNon and-iron metals iron or other metals are transported by otherFe transporters metals

Adapted from Thomine, S. and Vert, G. (2013). Iron transport in plants: better be safe than sorry. Curr. Opin. Plant Biol. 16: 322-327.

Flavodoxin fills the WT +Fld same role but does 2Fe-2S not require iron. Cyanobacteria and many algae can use flavodoxin (Fld) in place of ferredoxin Fld-expressing plants are (Fd) under iron Flavodoxin (Fld) more stress tolerant deficiency Ferredoxin (Fd)

Tognetti, V.B., Palatnik, J.F., Fillat, M.F., Melzer, M., Hajirezaei, M.-R., Valle, E.M. and Carrillo, N. (2006). Functional replacement of ferredoxin by a cyanobacterial flavodoxin in tobacco confers broad-range stress tolerance. Plant Cell. 18: 2035-2050.

There are three types of superoxide dismutases (SODs) encoded by different genes. The encoded proteins are Mg-SODs,

Fe-SODs or CuZn-SODs. Activity In Fe-limited Chlamydomonas reinhardtii , MnSOD3 expression and activity increase to compensate for

decreased FeSOD activity Protein

Reprinted from Pilon, M., Ravet, K. and Tapken, W. (2011). The biogenesis and physiological function of chloroplast superoxide dismutases. Biochim. Biophys. Acta. 1807: 989-998 with permission from Elsevier; Page, M.D., Allen, M.D., Kropat, J., Urzica, E.I., Karpowicz, S.J., Hsieh, S.I., Loo, J.A. and Merchant, S.S. (2012). Fe sparing and Fe recycling contribute to increased superoxide dismutase capacity in iron-starved Chlamydomonas reinhardtii. Plant Cell. 24: 2649-2665

Iron deficiency causes anemia

The World Health Organization estimates that two billion people Fe deficiency suffer micronutrient deficiencies

Zinc deficiency causes compromised immunity, decreased growth rate, and neurological effects including mental retardation

Zn deficiency

Maps from HarvestPlus based on WHO data

Reprinted from Masuda, H., Aung, M. and Nishizawa, N. (2013). Iron biofortification of rice using different transgenic approaches. Rice. 6: 40.

Increase sequestration in grain (ferritin, Post-harvest nicotianamine synthesis) fortification

Lower levels of antinutrients High-Zn and -Fe beans, (e.g., phytate) high-Zn rice, and high-Fe wheat are micronutrient- enriched crops Fertilize soil with nutrients Increase transporter activity

Increase production of phytosiderophores

Adapted from Stein, A. (2010). Global impacts of human mineral malnutrition. Plant Soil. 335: 133-154; Shahzad, Z., Rouached, H. and Rakha, A. (2014). Combating mineral malnutrition through iron and zinc biofortification of cereals. Comp. Rev. Food Sci Food Safety. 13: 329-346; Zhao, F.-J. and McGrath, S.P. (2009). Biofortification and phytoremediation. Curr. Opin. Plant Biol. 12: 373-380; Neil Palmer, CIAT

+Cu −Cu +Cu −Cu +Cu +Cu −Cu

Sommer, A.L. (1931) Copper as an essential for plant growth.PlantPhysiol.6: 339–45; Image © Ute Krämer (RUB), Josef Bergstein (MPI Golm), used with permission

Cu/Zn Superoxide Dismutase

Laccase / multicopper Plastocyanin Cytochrome c oxidase / oxidase ferroxidase

Ethylene receptor (in ER)

Images courtsy of Michael Leonard, Sabeeha Merchant, Somepics; Rozzychan

Cu(I) Entry Chaperone

Cu-metallochaperones have a “trafficking” role

Cu-metallothioneins have a “buffering“ role (like a sponge)

Reproduced from Banci, L., Bertini, I., McGreevy, K.S. and Rosato, A. (2010). Molecular recognition in copper trafficking. Natural Prod. Rep. 27: 695-710 with permission of the Royal Society of Chemistry

Cu-Chaperone Cu-Target • Copper chaperones interact closely with their delivery targets • Different copper-binding proteins usually have different chaperones

Cu is transferred directly from chaperone to target

Cu-Chaperone Cu-Target

Fe can also be shielded by chaperones but they are less well characterized than Cu chaperones Reproduced from Banci, L., Bertini, I., McGreevy, K.S. and Rosato, A. (2010). Molecular recognition in copper trafficking. Natural Prod. Rep. 27: 695-710 with permission of the Royal Society of Chemistry; see also O'Halloran, T.V. and Culotta, V.C. (2000). Metallochaperones, an intracellular shuttle service for metal ions. J. Biol. Chem. 275: 25057-25060..

To enter the lumen of the thylakoid, Chaperone copper has to cross three membranes, the inner and outer Target copper chloroplast envelopes and the transporter thylakoid membrane. PAA1 and PAA2 are copper transporters in the inner envelope and thylakoid membrane.

PCH1 is a chaperone that delivers Cu to PAA1, and in some plants it is translated from an alternatively spliced PAA1 transcript.

Reprinted from Blaby-Haas, C.E., Padilla-Benavides, T., Stübe, R., Argüello, J.M. and Merchant, S.S. (2014). Evolution of a plant-specific copper chaperone family for chloroplast copper homeostasis. Proc. Natl. Acad. Sci. USA. 111: E5480-E5487

COPT is a small COPT? family of high- YSL? affinity Cu influx PAA2 transporters Cu-NA PAA1 Copper chaperones Rhizosphere Root cell HMA? ER COPT ? OsHMA5 Leaf cell

Cu(I) FRO4

and Copper FRO5 Cu(II) chaperone Cu-NA? Xylem Copper reductase

Adapted from Burkhead, J.L., Gogolin Reynolds, K.A., Abdel-Ghany, S.E., Cohu, C.M. and Pilon, M. (2009). Copper homeostasis. New Phytol. 182: 799-816; See also Yruela, I. (2009). Copper in plants: acquisition, transport and interactions. Funct. Plant Biol. 36: 409-430; Krämer, U, and Clemens, S. (2006). Functions and homeostasis of zinc, copper, and nickel in plants. Molecular Biology of Metal Homeostasis and Detoxification. 14: 215-271. Deng, F., Yamaji, N., Xia, J. and Ma, J.F. (2013). A member of the Heavy Metal P-Type ATPase OsHMA5 is involved in xylem loading of copper in rice. Plant Physiol. 163: 1353-1362; Bernal, M., et al. (2012) Transcriptome sequencing identifies SPL7-regulated copper acquisition genes FRO4/ FRO5 and the copper dependence of iron homeostasis in Arabidopsis. Plant Cell 24: 738–761.

In Arabidopsis, when [Cu] is low, FSD1 (encoding FeSOD) is transcribed, as is miRNA398, which targets CSD and CCS mRNAs (switching off non- essential Cu proteins).

When [Cu] is high, FSD1 and miRNA398 are not transcribed, and CSD and CCS mRNAs are transcribed

replaced by cytochrome c6 (Fe-heme protein) when Cu is deficient

CRR = Copper response regulator -Cu CRR

Cyt c6 CYC6 CRR Fe

protease Hours since Cu addition -Cu 2 6 16 26 +Cu PC Plasto- cyanin Copper PC

Cyt c 6 Iron (Fe) Proteolysis

Kropat, J., Gallaher, S.D., Urzica, E.I., Nakamoto, S.S., Strenkert, D., Tottey, S., Mason, A.Z. and Merchant, S.S. (2015). Copper economy in Chlamydomonas: Prioritized allocation and reallocation of copper to respiration vs. photosynthesis. Proc. Natl. Acad. Sci. USA 112 : 2644-2651 ; Merchant, S., and Bogorad, L. (1986). Regulation by copper of the expression of plastocyanin and cytochrome c552 in Chlamydomonas reinhardi. Mol. Cell. Biol. 6: 462–469.

Zinc is deficient in 50% of the world’s agricultural soils Barley grown with and is recognized as the world’s most critical and without zinc micronutrient deficiency in crops. In humans, Zn deficiency contributes to 800,000 child deaths annually

Soil

Sommer, A.L., and Lipman, C.B. (1926). Evidence on the indispensable nature of zinc and boron for higher green plants. Plant Physiol. 1: 231–249. Alloway BJ. 2007: Zinc in Soils and Crop Nutrition. IZA Publications. International Zinc Association, Brussels

Photo credits: South Dakota State University; IPNI; Howard F. Schwartz, Colorado State University, Bugwood.org; IRRI

The ribosomal proteins of the large ribosomal subunit with Zn circled • Several ribosomal proteins are Zn- proteins. • As there are millions of ribosomes in a cell, they make up the largest pool of cellular Zn. • Some prokaryotes can switch to a non-Zn ribosomal protein during Zn deficiency

Reprinted from Klinge, S., Voigts-Hoffmann, F., Leibundgut, M., Arpagaus, S. and Ban, N. (2011). Crystal structure of the eukaryotic 60S ribosomal subunit in complex with initiation factor 6. Science. 334: 941-948 with permission from AAAS.

C H Zn stabilizes the Zn-finger domain. In this C C2H2 fold, two Cys and two His interact with H Zn. Some proteins have many Zn fingers Zn

TFIIIA (the first identified Zn-finger protein) bound to DNA Zn-fingers are particularly well suited for interactions with nucleic acids; several are DNA- or RNA-binding proteins

Knight, R. and Shimeld, S. (2001). Identification of conserved C2H2 zinc-finger gene families in the Bilateria. Genome Biology. 2: research0016.0011 - research0016.0018. Laity, J.H., Lee, B.M. and Wright, P.E. (2001). Zinc finger proteins: new insights into structural and functional diversity. Curr. Opin. Struct. Biol. 11: 39-46 with permission from Elsevier. Nolte, R.T., Conlin, R.M., Harrison, S.C. and Brown, R.S. (1998). Differing roles for zinc fingers in DNA recognition: Structure of a six-finger transcription factor IIIA complex. Proc. Natl. Acad. Sci. USA.. 95: 2938-2943.

Zn deficiency activates transcription factors that induce expression of Zn transporters for increased Zn uptake

Reprinted from Sinclair, S.A. and Krämer, U. (2012). The zinc homeostasis network of land plants. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 1823: 1553-1567 with permission from Elsevier.

Transcriptional response to Zn- -Zn deficiency requires a pair of transcription factors that have a metal-binding domain

Assunção, A.G.L., Herrero, E., Lin, Y.-F., Huettel, B., Talukdar, S., Smaczniak, C., Immink, R.G.H., van Eldik, M., Fiers, M., Schat, H. and Aarts, M.G.M. (2010). Arabidopsis thaliana transcription factors bZIP19 and bZIP23 regulate the adaptation to zinc deficiency. Proc. Natl. Acad. Sci. 107: 10296-10301. Reprinted from Assunção, A.G.L., Persson, D.P., Husted, S., Schjorring, J.K., Alexander, R.D. and Aarts, M.G.M. (2013). Model of how plants sense zinc deficiency. Metallomics. 5: 1110-1116 with permission from Royal Society of Chemistry.

The light-dependent Mn limitation interferes reaction that splits with O2 evolution water and releases

oxygen depends on a

Mn cluster

released

2 O

Mn Symptoms of Mn deficiency

MnSOD is the dominant SOD in mitochondria

Reprinted from Iwata, S. and Barber, J. (2004). Structure of photosystem II and molecular architecture of the oxygen-evolving centre. Curr. Opin. Struct. Biol. 14: 447-453 with permission from Elsevier; Cheniae, G.M. and Martin, I.F. (1969). Photoreaction of Manganese Catalyst in Photosynthetic Oxygen Evolution. Plant Physiol. 44: 351-360. http://fyi.uwex.edu/discoveryfarms/2011/06/soil-conditions-and-plant-analysis-for- micronutrient-crop-nutrition/. Page, M.D., Allen, M.D., Kropat, J., Urzica, E.I., Karpowicz, S.J., Hsieh, S.I., Loo, J.A. and Merchant, S.S. (2012). Fe sparing and Fe recycling contribute to increased superoxide dismutase capacity in iron-starved Chlamydomonas reinhardtii. Plant Cell. 24: 2649-2665

Reprinted from Vogt, L., Vinyard, D.J., Khan, S. and Brudvig, G.W. (2015). Oxygen-evolving complex of Photosystem II: an analysis of second-shell residues and hydrogen-bonding networks. Curr. Opin. Chem. Biol. 25: 152-158 with permission from Elsevier.

radicle

cotyledon

Mn (blue) accumulates in leaf mesophyll (photosynthetic cells)

radicle

cotyledon

Socha, A.L. and Guerinot, M.L. (2014). Mn-euvering manganese: the role of transporter gene family members in manganese uptake and mobilization in plants. Front. Plant Sci. 5: 106.

When Mn is low, the transporter When Mn is high, OsNramp3 OsNramp3 moves Mn into phloem is degraded and Mn is for transport to young tissues transported to mature tissues

Young Mature tissues tissues

OsNramp3

Image courtesy of J. Ma, adapted from Yamaji, N., Sasaki, A., Xia, J.X., Yokosho, K. and Ma, J.F. (2013). A node-based switch for preferential distribution of manganese in rice. Nat Commun. 4: 2442.

(Moco) 2- MoO4

1939 Mo was shown to be an essential micronutrient

The genes involved in pterin moiety are conserved across the domains of life

Arnon, D.I., and Stout, P.R. (1939). Molybdenum as an essential element for higher plants. Plant Physiol. 14: 599-602. Mendel, R.R. (2013). The molybdenum cofactor. J. Biol. Chem. 288: 13165-13172.

2- MoO4

MOT1, MOT2 Cytosol

Reversible 2- chelation MoO4 CNX Moco 2- 2- MoO4 resembles SO4 and pathway high-affinity transport takes MOT2 place through MOT1 and

MOT2, members of the 2- Vacuole Moco MoO4 sulfate-transporter family

Moco enzymes

Adapted from Tejada-Jimenez, M., Chamizo-Ampudia, A., Galvan, A., Fernandez, E. and Llamas, A. (2013). Molybdenum metabolism in plants. Metallomics. 5: 1191-1203.

Mo-dependent enzymes in Arabidopsis • Nitrate reductase (NR), key step in inorganic nitrogen assimilation • Sulfite oxidase (SO), detoxifes excess Moco sulfite (probably) Nitrate reductase • Aldehyde oxidase(s) (AO), last step in active site ABA biosynthesis • Xanthine dehydrogenase (XDH), purine catabolism and stress reactions

Reprinted by permission from Fischer, K., Barbier, G.G., Hecht, H.-J., Mendel, R.R., Campbell, W.H. and Schwarz, G. (2005). Structural basis of eukaryotic nitrate reduction: Crystal structures of the nitrate reductase active site. Plant Cell. 17: 1167-1179. Mendel, R.R. and Hänsch, R. (2002). Molybdoenzymes and molybdenum cofactor in plants. J. Exp. Bot. 53: 1689-1698.See also Schwarz, G., Mendel, R.R., and Ribbe, M.W. (2009). Molybdenum cofactors, enzymes and pathways. Nature 460: 839 – 847.

FeMoco is used by the bacterial enzyme nitrogenase. This enzyme is needed for symbiotic nitrogen fixation that occurs in N2-fixing plant nodules

FeMoco

N N2 2 Moco Nitrogenase Note that FeMoco NH + 4 and Moco are totally different NH + 4 structures

From: Buchanan, B.B., Gruissem, W. and Jones, R.L. (2000) Biochemistry and Molecular Biology of Plants. American Society of Plant Physiologists.MacLeod, K.C. and Holland, P.L. (2013). Recent developments in the homogeneous reduction of dinitrogen by molybdenum and iron. Nat Chem. 5: 559-565; See also Schwarz, G., Mendel, R.R., and Ribbe, M.W. (2009). Molybdenum cofactors, enzymes and pathways. Nature 460: 839 – 847.

The requirement for Ni was demonstrated recently 1975 1983 1987 In Ni-deficient soybean, Ni-deficient barley The enzyme urease urea accumulation causes seeds cannot requires Ni tissue death germinate

Urease

Ni in urease active site Other Ni-dependent Germination % proteins are suspected but Ni (ng/g) have not been identified

Carter, E.L., Flugga, N., Boer, J.L., Mulrooney, S.B. and Hausinger, R.P. (2009). Interplay of metal ions and urease. Metallomics. 1: 207-221; Dixon, N.E., Gazzola, C., Blakeley, R.L. and Zerner, B. (1975). Jack bean urease (EC 3.5.1.5). Metalloenzyme. Simple biological role for nickel. J. Am. Chem. Soc.. 97: 4131-4133. Ragsdale, S.W. (2009). Nickel-based Enzyme Systems. J. Biol. Chem. 284: 18571-18575; Eskew, D.L., Welch, R.M. and Caru, E.E. (1983). Nickel: An essential micronutrient for legumes and possibly all higher plants. Science. 222: 621-623 with permission from AAAS; Brown, P.H., Welch, R.M. and Cary, E.E. (1987). Nickel: A micronutrient essential for higher plants. Plant Physiol. 85: 801-803.

Alyssum lesbiscum, a nickel Ni uses many of the same transporters as Fe, and hyperaccumulator shows greatly its toxicity may be due in part to competition with Fe elevated levels of the amino acid histidine which is able to chelate Ni

Vacuole Ni ZIP Fe

Ni-NA YSL Fe-NA IREG2

NRAMP ?

Adapted from Tejada-Jiménez, M., Galván, A., Fernández, E. and Llamas, Á. (2009). Homeostasis of the micronutrients Ni, Mo and Cl with specific biochemical functions. Curr. Opin. Plant Biol. 12: 358-363; Reprinted from Kramer, U., Cotter-Howells, J.D., Charnock, J.M., Baker, A.J.M. and Smith, J.A.C. (1996). Free histidine as a metal chelator in plants that accumulate nickel. Nature. 379: 635-638 by permission.

© 2015 American Society of Plant Biologists Plants able to tolerate high levels or Ni can be found on serpentine soils Serpentine soils: Low Ca / Mg ratio, low abundance macronutrients, high abundance metals including Ni. Plants here are often specialized to tolerate high Ni

Many Ni-tolerant species have evolved in New Caledonia

Serpentine soils in the Klamath Mountain area in Northern California / Southern Oregon

New Caledonia Plants; Miguel Vieira

© 2015 American Society of Plant Biologists Several hundred species are known to be nickel hyperaccumulators Two of the most widely studied are Noccaea caerulescens (formerly Thlaspi caerulescens; left) and Alyssum murale (right). Both are being explored for their potential in extracting metals from soils, and to learn about the mechanisms of metal hyperaccumulation

Konrad Lackerbeck; Jerzy Opioła

© 2015 American Society of Plant Biologists Metal tolerance and metal hyperaccumulation Defined as excluding metals from shoot. Some excluders accumulate metals in roots Metal Metal tolerant Metal tolerant non- Metal tolerant Hyperaccumulating sensitive by exclusion hyperaccumulating hyperaccumulating plants tend to store the metal in the shoot vacuoles, where it often contributes to defense against pathogens and herbivores

Reprinted from Lin, Y.-F. and Aarts, M.M. (2012). The molecular mechanism of zinc and cadmium stress response in plants. Cellular and Molecular Life Sciences. 69: 3187-3206 With kind permission from Springer Science and Business Media; see also Rascio, N. and Navari-Izzo, F. (2011). Heavy metal hyperaccumulating plants: How and why do they do it? And what makes them so interesting? Plant Science. 180: 169-181..

A hyperaccumulator actively acquires metals

A metal tolerant plant can survive high levels of metal in the soil A normal plant dies if

Metal in in plant Metal there is too much metal in the soil

Death Metal in soil

Goolsby, E.W. and Mason, C.M. (2015). Toward a more physiologically and evolutionarily relevant definition of metal hyperaccumulation in plants. Front. Plant Sci. 6: 33. Adapted from van der Ent, A., Baker, A.M., Reeves, R., Pollard, A.J. and Schat, H. (2013). Hyperaccumulators of metal and metalloid trace elements: Facts and fiction. Plant Soil. 362: 319-334.

• Metals that can be hyperaccumulated by various plants include: Nickel, zinc, cobalt, chromium, molybdenum, cadmium, arsenic, and selenium

These fascinating plants caught the eye of the famous artist / illustrator Maki Naro, who let us share his illustrations

Drawing by Maki Naro, reprinted with permission

© 2015 American Society of Plant Biologists A number of metal transporters Transporters demonstrated are more highly expressed in to contribute to Zn hyperaccumulators including hyperaccumulation Arabidopsis halleri and Noccaea caerulescens. Elevated levels of chelators such as nicotianamine are also often observed in hyperaccumulators

Arabidopsis halleri

Noccaea caerulescens (Alpine pennycress)

Reprinted from Hanikenne, M. and Nouet, C. (2011). Metal hyperaccumulation and hypertolerance: a model for plant evolutionary genomics. Curr. Opin. Plant Biol. 14: 252-259 with permission from Elsevier. HermannSchachner ; Wikipedia

Hyperaccumulating plants can be used to remove metals from soils (phytoremediation) or to concentrate and use the metals (phytomining or metal farming)

Sap containing ~ 16% nickel exuding from a cut branch of the tree Phyllanthus balgooyi.

Favas, P.J.C., Pratas, J., Varun, M., D’Souza, R., and Paul, M.S. (2014). Phytoremediation of Soils Contaminated with Metals and Metalloids at Mining Areas: Potential of Native Flora, Environmental Risk Assessment of Soil Contamination, Dr. Maria C. Hernandez Soriano (Ed.), ISBN: 978-953-51-1235-8, InTech, DOI: 10.5772/57469.; Photo by Antony van der Ent, used by permission of AusIMM Bulletin.

Cadmium interferes with zinc uptake and Arsenate [As(V)] interferes activities, affects copper Al inhibits with phosphate uptake and homeostasis, and root growth function, and arsenite moves through iron [As(III)] can move through transporters silicate transporters

Arsenic is abundant, toxic and a major human health concern throughout south and south-east Asia where As naturally occurs in soils and groundwaters

Reprinted from Brammer, H. and Ravenscroft, P. (2009). Arsenic in groundwater: A threat to sustainable agriculture in South and South-east Asia. Environment International. 35: 647- 654 with permission from Elsevier; see also Hasanuzzaman, M., Nahar, K., Hakeem, K.R., Öztürk, M., and Fujita, M. (2015). Arsenic toxicity in plants and possible remediation. In Soil Remediation and Plants, K.Hakeem, M. Sabir, M. Öztürk and A.Murmet, eds (Amsterdam: Elsevier), pp 433 -501.

© 2015 American Society of Plant Biologists In severely arsenic-affected Eating As- areas, there are contaminated several routes to rice grain human exposure Burning rice straw leads to volatilization and inhalation

Drinking water As in soil and from shallow wells groundwater that are As moves into plants contaminated

Reprinted from Rahman, M.A., Hasegawa, H., Mahfuzur Rahman, M., Mazid Miah, M.A. and Tasmin, A. (2008). Arsenic accumulation in rice (Oryza sativa L.): Human exposure through food chain. Ecotoxicology and Environmental Safety. 69: 317-324 with permission from Elsevier.

© 2015 American Society of Plant Biologists Rice is particularly prone to arsenic uptake accumulation Inorganic As is mainly found as arsenate As(V) and arsenite As(III). In As(OH)3 anaerobic or flooded soils such as rice paddies, As(III) predominates As(V) ↑

As(III) As(OH)3

In rice, Lsi1 and Lsi2 transporters are As(OH) highly expressed for efficient Si uptake, 3 and As(III) exploits this pathway

Ma, J.F., Yamaji, N., Mitani, N., Xu, X.-Y., Su, Y.-H., McGrath, S.P. and Zhao, F.-J. (2008). Transporters of arsenite in rice and their role in arsenic accumulation in rice grain. Proc. Natl. Acad. Sci. USA. 105: 9931- 9935; Adapted from Zhao, F.-J., McGrath, S.P. and Meharg, A.A. (2010). Arsenic as a food chain contaminant: Mechanisms of plant uptake and metabolism and mitigation strategies. Annu. Rev. Plant Biol. 61: 535- 559 and Verbruggen, N., Hermans, C. and Schat, H. (2009). Mechanisms to cope with arsenic or cadmium excess in plants. Curr. Opin. Plant Biol. 12: 364-372.

Increasing synthesis of phytochelatins, increased sequestration in the vacuole, and increased efflux can contribute to lower As levels in the rice grain

As(V) Arsenate PC reductase synthase Adding As(V) competing P As(III) Phytochelatin 2- or Si to soil PO4 interferes with As(III) As(III)-PC As uptake As(III)

Si Xylem

Song, W.-Y., Yamaki, T., Yamaji, N., Ko, D., Jung, K.-H., Fujii-Kashino, M., An, G., Martinoia, E., Lee, Y. and Ma, J.F. (2014). A rice ABC transporter, OsABCC1, reduces arsenic accumulation in the grain. Proc. Natl. Acad. Sci. USA. 111: 15699-15704;

Cd is usually mixed with zinc and released during the mining process

In the mid-20th century widespread cadmium poisoning occurred Tobacco accumulates Cd, downstream of mining facilities in so smoking significantly Toyama prefecture, Japan increases your intake of Cd P-containing fertilizers are often It was known as itai-itai Cd-contaminated (ouch-ouch) disease after the painful symptoms, which include bone decalcification, Ni-Cd lung and kidney disease batteries

JJ Harrison; Kanazawa Med

Also CAX cation / proton exchangers

Reprinted from Clemens, S., Aarts, M.G.M., Thomine, S. and Verbruggen, N. (2013). Plant science: the key to preventing slow cadmium poisoning. Trends Plant Sci. 18: 92-99 with permission from Elsevier.

© 2015 American Society of Plant Biologists Breeding for altered transporter activities leads to low-Cd rice Altering activities of several different transporters including OsNramp5, LCT1 and HMA3 contributes to low-Cd rice grains

Control (L) and low-Cd (right) grains stained with QAI, which turns purple when it reacts with Cd

Uraguchi, S. and Fujiwara, T. (2012). Cadmium transport and tolerance in rice: perspectives for reducing grain cadmium accumulation. Rice. 5: 5; Uraguchi, S., et al. (2011). Low-affinity cation transporter (OsLCT1) regulates cadmium transport into rice grains. Proc. Natl. Acad. Sci. 108: 20959-20964. See also Uraguchi, S. and Fujiwara, T. (2013). Rice breaks ground for cadmium-free cereals. Curr. Opin. Plant Biol. 16: 328-334. Ishikawa, S., et al. (2012). Ion-beam irradiation, gene identification, and marker-assisted breeding in the development of low-cadmium rice. Proc. Natl. Acad. Sci. USA 109: 19166-19171.

Aluminum is the most abundant metal in the Earth’s crust. At neutral pH it is bound into Low pH Al3+ insoluble complexes. Acidic soils release Al3+ which arrests Soluble Aluminum root growth in sensitive plants sensitive Solid

Aluminum tolerant

Aluminum tolerance is genetically determined

Much of the world’s soil is strongly (pH ≤ 4.5) or moderately acidic (pH 4.6 – 5.5)

Brunner, I. and Sperisen, C. (2013). Aluminium exclusion and aluminium tolerance in woody plants. Front. Plant Sci. 4: 172; Delhaize, E. and Ryan, P.R. (1995). Aluminum Toxicity and Tolerance in Plants. Plant Physiol. 107: 315-321.

Aluminum Aluminum Al-tolerant varieties excrete sensitive tolerant much more organic acid than sensitive varieties

Al3+ Al3+ Al3+ Al3+ Al3+ 3+ Al3+ Al Al3+

Organic acids form complexes with Al and Al prevent its uptake

Delhaize, E., Ryan, P.R. and Randall, P.J. (1993). Aluminum Tolerance in Wheat (Triticum aestivum L.) (II. Aluminum-Stimulated Excretion of Malic Acid from Root Apices). Plant Physiol. 103: 695-702.

Barley engineered with a malate transporter shows increased Al tolerance as compared to wild type

Schroeder, J.I., Delhaize, E., Frommer, W.B., Guerinot, M.L., Harrison, M.J., Herrera-Estrella, L., Horie, T., Kochian, L.V., Munns, R., Nishizawa, N.K., Tsay, Y.-F. and Sanders, D. (2013). Using membrane transporters to improve crops for sustainable food production. Nature. 497: 60-66 by permission of Nature Publishing Group.

Organic acid Transcriptional secretion responses

Changes in Vacuolar mitochondrial sequestration metabolism

ROS accumulation and detoxification

Brunner, I. and Sperisen, C. (2013). Aluminium exclusion and aluminium tolerance in woody plants. Front. Plant Sci. 4: 172; See also Nunes-Nesi, A., Brito, D.S., Inostroza-Blancheteau, C., Fernie, A.R. and Araújo, W.L. (2014). The complex role of mitochondrial metabolism in plant aluminum resistance. Trends Plant Sci. 19: 399-407. See also Delhaize, E., Ma, J.F. and Ryan, P.R. (2012). Transcriptional regulation of aluminium tolerance genes. Trends Plant Sci. 17: 341-348..

in rice FRDL4 exports citrate which immobilizes external Al ART1 is an Al-induced transcription factor that Nrat1 is an upregulates Al uptake Al tolerance transporter genes (In Arabidopsis, START has the same ALS1 function) imports Al into the vacuole

Image provided courtesy of J. Ma from Ma, J., Chen, Z. and Shen, R. (2014). Molecular mechanisms of Al tolerance in gramineous plants. Plant Soil. 381: 1-12; see references therein

Aluminum hyperaccumulating species occur in at least 45 plant families

The blue color of hydrangeas comes from metalloanthocyanin pigments Melastoma malabathricum that form when pH is lowered Camellia sinensis and Al uptake is facilitated

Yoshida, K., Mori, M. and Kondo, T. (2009). Blue flower color development by anthocyanins: from chemical structure to cell physiology. Natural Product Reports. 26: 884-915; Schreiber, H., Jones, A., Lariviere, C., Mayhew, K. and Cain, J. (2011). Role of aluminum in red-to-blue color changes in Hydrangea macrophylla sepals. BioMetals. 24: 1005-1015. Tu7uh; AxelBoldt; Jansen, S., Broadley, M., Robbrecht, E. and Smets, E. (2002). Aluminum hyperaccumulation in angiosperms: A review of its phylogenetic significance. Bot. Rev. 68: 235-269; Heather Coleman.

Boron is found in soils and also mined Boron mine Boron was identified as an for industrial use essential micronutrient in 1923

Other uses include • Borosilicate glass • Laundry soap • Insecticide

1:5000 Boric Acid 1:50,000 Boric Acid No Boric Acid

Reprinted from Warington, K. (1923). The effect of boric acid and borax on the broad bean and certain other plants. Ann. Bot. 37: 629-672 with permission from Oxford University Press; Preiselbeere; Marcin Wichary .

Boron can crosslink In plant cell walls, B crosslinks complex polysaccharides called Rhamnogalacturonan II (RG-II), made up of a molecules by making diester backbone and four side chains bridges between them Rhamnogalacturonan II Backbone - H3BO3 B(OH)4 HO HO − OH Two RG-II B OH B HO HO OH crosslinked by boron

HO − O C C O − O C B B HO O C C O O C

Adapted from Bolaños, L., Lukaszewski, K., Bonilla, I. and Blevins, D. (2004). Why boron? Plant Physiol. Biochem. 42: 907-912; O'Neill, M.A., Eberhard, S., Albersheim, P. and Darvill, A.G. (2001). Requirement of borate cross-linking of cell wall Rhamnogalacturonan II for Arabidopsis growth. Science. 294: 846-849 with permission from AAAS. Complex Carbohydrate Research Center

© 2015 American Society of Plant Biologists Boron influx mutants have developmental and cell wall defects Loss of function mutant of B transporter tsl has cell wall and developmental defects TSL encodes a NIP boron influx transporter. The mutant phenotype is rescued by added boron

Chromatograph showing size distribution of wall components from wildtype (top) and tsl mutant tissues; the mutant shows a cross-linking deficiency (decrease in RG-II dimer).

Durbak, A.R., et al. (2014). Transport of boron by the tassel-less1 aquaporin is critical for vegetative and reproductive development in maize. Plant Cell. 26: 2978-2995; See also Leonard, A., et al. (2014). tassel-less1 encodes a boron channel protein required for inflorescence development in maize. Plant Cell Physiol. 55: 1044-1054; Takano, J., Miwa, K. and Fujiwara, T. (2008). Boron transport mechanisms: collaboration of channels and transporters. Trends Plant Sci. 13: 451-457 with permission from Elsevier.

RTE encodes BOR1, a boron efflux transporter. Loss-of-function is thought to prevent B from reaching the shoot in the Boron Boron xylem transpiration stream influx efflux

The phenotype is rescued with added boron

Chatterjee, M., Tabi, Z., Galli, M., Malcomber, S., Buck, A., Muszynski, M. and Gallavotti, A. (2014). The boron efflux transporter ROTTEN EAR is required for maize inflorescence development and fertility. Plant Cell. 26: 2962-2977. Takano, J., Miwa, K. and Fujiwara, T. (2008). Boron transport mechanisms: collaboration of channels and transporters. Trends Plant Sci. 13: 451-457 with permission from Elsevier.

Highly expressed

Poorly expressed

Non-functional

Different alleles of Bo1 B transporter confer more or less tolerance to high boron (allele structure and root length figure use same colors). Highly expressed alleles (red) confer greater B tolerance than poorly expressed (green) or non- functional alleles.

Reprinted from Pallotta, M., Schnurbusch, T., Hayes, J., Hay, A., Baumann, U., Paull, J., Langridge, P. and Sutton, T. (2014). Molecular basis of adaptation to high soil boron in wheat landraces and elite cultivars. Nature. 514: 88-91 with permission from Nature Publishing Group.

Orange shows countries or regions where B toxicity has been identified

Red allele is tolerant Black diamonds indicate predicted sources of the tolerance alleles, with proposed dispersion shown by black arrows. Red circles show countries where modern cultivars carrying tolerance alleles have been found.

High levels of boron promote the ubiquitination of BOR and its internalization to the multi- vesicular body (MVB) and vacuole.

When external B levels are high, this transporter internalization prevents continued transport of boron to the shoot, limiting boron toxicity

Kasai, K., Takano, J., Miwa, K., Toyoda, A. and Fujiwara, T. (2011). High boron-induced ubiquitination regulates vacuolar sorting of the BOR1 borate transporter in Arabidopsis thaliana. J. Biol. Chem. 286: 6175-6183; See also Zelazny, E. and Vert, G. (2014). Plant nutrition: Root transporters on the move. Plant Physiol. 166: 500-508.

© 2015 American Society of Plant Biologists Silicon is essential for some plants, beneficial for many Silicon (Si) is the In 1969 Chen and second most abundant Equisetum arvense Lewis showed Si to be element in the earth’s essential for growth of crust after oxygen the common horsetail, Equisetum arvense It is often found as silica SiO (insoluble 2 Diatoms form cell walls from silicon quartz sand), and in biological systems as silicic acid Si(OH)4

See Chen, C.-h., and Lewin, J. (1969). Silicon as a nutrient element for Equisetum arvense. Can. J. Bot. 47: 125-131; Painting by Carl Axel Magnus Lindman; MPF. Norris, D.J. (2007). Materials science: Silicon life forms. Nature 446: 146 – 147 with permission from Nature.

Powdery mildew on control plant

Plant treated with Si shows resistance

Reprinted from Ma, J.F. and Yamaji, N. (2006). Silicon uptake and accumulation in higher plants. Trends Plant Sci. 11: 392-397 with permission from Elsevier; Heckman, J. (2013) Silicon: A beneficial substance. Better Crops 97: 14 – 16.

© 2015 American Society of Plant Biologists Rice grown with low Si is susceptible to herbivores & pathogens The mechanisms of Si-conferred resistance remain unclear: • Physical barrier? • Enhancement of defense responses? • Priming of defense responses?

Silicon deposits in plant cells are called phytoliths (plant stones) and may protect against herbivory

Reprinted from Ma, J.F. and Yamaji, N. (2006). Silicon uptake and accumulation in higher plants. Trends Plant Sci. 11: 392-397 and Cooke, J. and Leishman, M.R. (2011). Is plant ecology more siliceous than we realise? Trends Plant Sci. 16: 61-68 with permission from Elsevier. See also Vivancos, J., Labbé, C., Menzies, J.G. and Bélanger, R.R. (2015). Silicon-mediated resistance of Arabidopsis against powdery mildew involves mechanisms other than the salicylic acid (SA)- dependent defence pathway. Mol. Plant Pathol. In press Van Bockhaven, J., De Vleesschauwer, D. and Höfte, M. (2013). Towards establishing broad-spectrum disease resistance in plants: silicon leads the way. J. Exp. Bot. 64: 1281-1293.

© 2015 American Society of Plant Biologists Uptake and transport of silicon in rice; Arsenic uses the same pathway Si passes through the exo- and endodermis through the action of a pair of silicon transporters, Lsi1 and Lsi2 Lsi1 Lsi2

exodermis

Image provided by J. Ma. See Mitani, N., Chiba, Y., Yamaji, N. and Ma, J.F. (2009). Identification and characterization of maize and barley Lsi2-Like silicon efflux transporters reveals a distinct silicon uptake system from that in rice. Plant Cell. 21: 2133-2142. Ma, J.F., Yamaji, N., Mitani, N., Xu, X.-Y., Su, Y.-H., McGrath, S.P. and Zhao, F.-J. (2008). Transporters of arsenite in rice and their role in arsenic accumulation in rice grain. Proc. Natl. Acad. Sci. USA. 105: 9931-9935; see also Van Bockhaven, J., De Vleesschauwer, D. and Höfte, M. (2013). Towards establishing broad-spectrum disease resistance in plants: silicon leads the way. J. Exp. Bot. 64: 1281-1293; See Ma, J.F., Tamai, K., Yamaji, N., Mitani, N., Konishi, S., Katsuhara, M., Ishiguro, M., Murata, Y. and Yano, M. (2006). A silicon transporter in rice. Nature. 440: 688-691; Ma, J.F., Yamaji, N., Mitani, N., Tamai, K., Konishi, S., Fujiwara, T., Katsuhara, M. and Yano, M. (2007). An efflux transporter of silicon in rice. Nature. 448: 209-212.

Cl in soil comes from rain, sea spray or the application of KCl,

CaCl2, or MgCl2 in fertilizers

Major cellular anions Chlorine (Cl) is taken up as Chloride Cl− chloride (Cl-) and − its primary role is Nitrate NO3 as a negatively charged anion Malate + Cl - Cl

Broyer, T.C., Carlton, A.B., Johnson, C.M., and Stout, P.R. (1954). Chlorine, a micronutrient element for higher plants. Plant Physiol. 29: 536 -532; Chloride deficiency photo used by permission of R.E. Engel.

Guard cells are one of the OPEN CLOSING most intensely studied cells. Stomatal opening occurs V-ATPase Proton PM-H+ when proton pumps energize -ATPase H+ the membrane and K+ and Cl- pumps H+ move into the vacuole, raising V-PPase cell turgor + H Cl- Cl- H+ Cl- H+ Chloride transporters Cl- Cl- K+ K+ + K K+

Potassium transporters

Eupachlorin acetate, isolated from Eupatorium rotundifolium, is a Cl-containing sesquiterpenoid with cytotoxic properties

Cl is the normal anion in the oxygen- evolving complex. In some studies other anions can substitute, although photosynthetic efficiency may be lowered

Reprinted from Iwata, S. and Barber, J. (2004). Structure of photosystem II and molecular architecture of the oxygen-evolving centre. Curr. Opin. Struct. Biol. 14: 447-453 with permission from Elsevier Guskov, A., Kern, J., Gabdulkhakov, A., Broser, M., Zouni, A. and Saenger, W. (2009). Cyanobacterial photosystem II at 2.9-A resolution and the role of quinones, lipids, channels and chloride. Nat Struct Mol Biol. 16: 334-342 with permission. Engvild, K.C. (1986). Chlorine-containing natural compounds in higher plants. Phytochemistry. 25: 781-791; USDA

For many plants, the detrimental effects of salinity are attributed Cl- exclusion mainly to Na+, but a few including can contribute soybean and citrus are particularly to Cl- tolerance sensitive to excess Cl-

Henderson, S.W., Baumann, U., Blackmore, D.H., Walker, A.R., Walker, R.R., and Gilliham, M. (2014). Shoot chloride exclusion and salt tolerance in grapevine is associated with differential ion transporter expression in roots. BMC Plant Biol. 14: 273.See also Teakle, N.L. and Tyerman, S.D. (2010). Mechanisms of Cl- transport contributing to salt tolerance. Plant Cell Environ. 33: 566-589 and Jossier, M., Kroniewicz, L., Dalmas, F., Le Thiec, D., Ephritikhine, G., Thomine, S., Barbier- Brygoo, H., Vavasseur, A., Filleur, S. and Leonhardt, N. (2010). The Arabidopsis vacuolar anion transporter, AtCLCc, is involved in the regulation of stomatal movements and contributes to salt tolerance. Plant J. 64: 563-576. USDA, USDA

© 2015 American Society of Plant Biologists Selenium is an essential micronutrient for animals Selenium deficiency 25 human genes encode selenoproteins, in which affects many systems the “21st amino acid” Se-Cys (abbreviated U) is and can cause death incorporated into the polypeptide Thyroid

Muscle Heart

Reproductive tissues

Too much Se in the diet is also bad!

Lu, J. and Holmgren, A. (2009). Selenoproteins. J. Biol. Chem. 284: 723-727; Labunskyy, V.M., Hatfield, D.L. and Gladyshev, V.N. (2014). Selenoproteins: Molecular pathways and physiological roles. Physiol. Rev. 94: 739-777. Hatfield, D.L., Tsuji, P.A., Carlson, B.A. and Gladyshev, V.N. (2014). Selenium and selenocysteine: roles in cancer, health, and development. Trends Biochem. Sci. 39: 112-120.

© 2015 American Society of Plant Biologists The amount of Se in soils has a direct effect on dietary levels Dark = High Keshan Pink = High Light = Low County Very low Se levels in northeast China Gray = Low contributed to many deaths from Se- deficiency, aka Keshan disease.

Wheat grain Se Western Australia 0.001 mg / kg concentration varies Serbia 0.028 mg / kg China 0.01 – 0.6 mg / kg globally and depends US (Average) 0.2 – 0.6 on soil Se levels South Dakota 30 mg / kg

Reprinted from Blazina, T., Sun, Y., Voegelin, A., Lenz, M., Berg, M. and Winkel, L.H.E. (2014). Terrestrial selenium distribution in China is potentially linked to monsoonal climate. Nat Commun. 5: 4717 by permission from Nature Publishing Group. See also Lyons, G., Stangoulis, J. and Graham, R. (2003). High-selenium wheat: biofortification for better health. Nutrit. Res. Rev. 16: 45-60. Zhu, Y.-G., Pilon-Smits, E.A.H., Zhao, F.-J., Williams, P.N. and Meharg, A.A. (2009). Selenium in higher plants: understanding mechanisms for biofortification and phytoremediation. Trends Plant Sci. 14: 436-442. USGS

Selenate Selenocysteine Selenocysteine can be Selenate (SeO 2-) is the major 4 (SeCys) introduced into selenoproteins assimilated form in most or converted into other forms plants. It resembles sulfate including volatile forms and is taken up through 2- sulfate SO4 transporters

Cystseine Sulfate (Cys)

Uptake as selenate Manipulation of these [Se(VI)] through sulfate pathways can contribute to transporters or as either biofortification and selenite [Se(IV)] through phytoremediation efforts; phosphate transporters for example, increasing expression of APS or SMT leads to increased accumulation of Se Se is incorporated into organic form through S assimilation pathway

Reprinted from Malagoli, M., Schiavon, M., Dall'Acqua, S. and Pilon-Smits, E.A.H. (2015). Effects of selenium biofortification on crop nutritional quality. Front. Plant Sci. 6: 280; Zhu, Y.-G., Pilon-Smits, E.A.H., Zhao, F.-J., Williams, P.N. and Meharg, A.A. (2009). Selenium in higher plants: understanding mechanisms for biofortification and phytoremediation. Trends Plant Sci. 14: 436-442. Van Hoewyk, D. (2013). A tale of two toxicities: malformed selenoproteins and oxidative stress both contribute to selenium stress in plants. Ann. Bot. 112: 965-972.

• Plants require small amounts of micronutrients for viability • Six essential micronutrients are d-block metals needed for catalysis or structure • B and Si function largely for structure, and Cl for charge and electrical balance • Al, Cd and As are non-essential, toxic elements Non-metals

Metalloids Metals D-block Essential micronutrients

Non-essential toxic elements (examples)

www.plantcell.org/cgi/doi/10.1105/tpc.109.tt1009

© 2015 American Society of Plant Biologists Transporters and chelators contribute to homeostasis Micronutrient homeostasis requires: • Membrane transporters • Small molecule metal chelators • Transporters for metal chelators • Metallochaperone proteins • Regulation by transcription, mRNA and protein stability, and protein subcellular localization

Many genes contributing to micronutrient homeostasis have been identified, paving the way for their use in breeding strategies

© 2015 American Society of Plant Biologists The study of plant micronutrients helps ensure: 1) Optimal crop yields, particularly in nutrient-poor soil, 2) Adequate dietary Cd iron and zinc so that children and adults can thrive, and 3) Helps protect people from the toxic effects of arsenic and cadmium

IRRI; CIAT; Water.org; Centre for Food Safety, Government of Hong Kong