Plant Nutrition – Micronutrients and Metals

Non-metals
Essential
Metalloids micronutrients
Metals
D-block metals

Non-essential
toxic elements
(examples)

Essential for
animals,
beneficial for
plants
www.plantcell.org/cgi/doi/10.1105/tpc.109.tt1009
 Outline

•  Introduction to micronutrients The hydroponics system developed by Hoagland and Arnon

for the characterization of micronutrients

•  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
Dennis Hoagland and colleagues developed a soil-
•  Summary and ongoing research 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.
 Micronutrients are essential in at least one plant
taxon
“An element is not
considered essen.al unless Or… “the plant can be so severely deficient in
a deficiency of it makes it the element that it exhibits abnormalities
impossible for the plant to in growth, development, or reproduction, i.e.
complete its life cycle” "performance," compared to plants with a
-Arnon and Stout, 1939 Mn lower deficiency”
B -Epstein and Bloom, 2003
Zn
Cu
Fe Mo Cl 1969 Ni
1920 - 1954 Si 1987
1860
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).
 Nutrient availability in soil is highly dependent on soil
pH

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

Fe(OH)3 + 3H+ Fe3+ + 3H2O

Strongly Mildly
Insoluble Soluble acidic alkaline
 Most micronutrients have a narrow optimal
concentration range

Essential nutrient Non-essential element

Optimal
range

Deficiency Luxury (plants

(plants can can store or
adjust) detoxify)

Limitation Toxicity Tolerance Toxicity

Plant dry weight

Plant dry weight

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.
Essential micronutrients are normally found in small
amounts
Element Biologically Concentra9on 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.
Metal micronutrients: What’s so special about
metals?

Metal micronutrients

Metals
D-block metals
 Most metal micronutrients are important cofactors
for enzymes

Enzymes that use Fe, Cu or Mo

Bar height = % of cellular proteins are mainly oxidoreductases
Macro- requiring element as cofactor (blue bars)
nutrients

Micronutrients

Enzymes that use Mn, or Zn

have more diverse functions:
Yellow= transferases; purple=
hydrolase; red= lyase; green=
isomerase; grey= ligase

Waldron, K.J., Rutherford, J.C., Ford, D. and Robinson, N.J. (2009). Metalloproteins and metal sensing. Nature. 460: 823-830.
 Many metal nutrients are redox active: necessary but
dangerous

e− Fenton reaction
About ¼ of cellular proteins
are metalloproteins (not M(n)+ + 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)
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
Fe(OH)3 + 3H+ Fe3+ + 3H2O
Insoluble Soluble
citrate
Iron citrate

Banci, L., Bertini, I., McGreevy, K.S. and Rosato, A. (2010). Molecular recognition in copper trafficking. Natural Prod. Rep. 27: 695-710
 Siderophores: Small metal-binding molecules to
facilitate uptake

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 is Fe-enterobactin
imported into the cell

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.
 Metals chelators: nicotianamine, phytosiderophores
and others
COOH
3x The enzyme In grasses, phytosiderophores
COOH COOH COOH
nicotiananamine are produced from NA, and
S+ NH2 N NH NH2
synthase (NAS) released from the plant into the
makes Nicotianamine
Adenosyl soil to enhance metal uptake
nicotianamine
(NA) from three Metal-PS
complex COOH COOH COOH
molecules of S-
COOH COOH COOH Yellow= metal
adenosyl Red = oxygen N NH OH
methionine Blue = nitrogen
N NH 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
the plant Citrate O
O−
Metal-NA N
complex + N
Ni H2
O
Purple = metal Metal H2 O−
Ni(His)
N
Red = oxygen HN
Blue = nitrogen Fe(III)-citrate

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 and metallothioneins are sulfur-
containing metal binders

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: Abundant, important, and largely insoluble

Iron is the fourth most

abundant element in
Oxygenic photosynthesis, beginning ~ 2.5 billion years
the earth’s crust ago, rendered Fe largely oxidized and insoluble. For
most cells, obtaining sufficient Fe is a challenge
Na Mg Rest
K
Fe Ca Fe(II) or Fe2+ Oxygen Fe(III) or Fe3+
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.
 Photosynthetic and respiratory electron transport
chains require Fe
Or flavodoxin (Cu)

Respiratory
electron
transport in
Or plastocyanin (Cu)
mitochondria

Photosynthetic
electron
transport in
chloroplasts

Blaby-Haas, C.E. and Merchant, S.S. (2013). Iron sparing and recycling in a compartmentalized cell. Curr. Opin. Microbiol. 16: 677-685
 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 ethylene FeSOD, found in

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

Balk, J. and Schaedler, T.A. (2014). Iron cofactor assembly in plants. Annu. Rev. Plant Biol. 65: 125-153; 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.
 Copper: Critical for aerobic life

+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)
 Copper proteins are involved in electron transport
and other rxns

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

 Cells have essentially no “free” copper; it is entirely
bound

Cu(I) Entry
Chaperone

Cu-metallochaperones
have a “trafficking” role

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

Banci, L., Bertini, I., McGreevy, K.S. and Rosato, A. (2010). Molecular recognition in copper trafficking. Natural Prod. Rep. 27: 695-710
 Cells have essentially no “free” copper; it is entirely
bound

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

Banci, L., Bertini, I., McGreevy, K.S. and Rosato, A. (2010). Molecular recognition in copper trafficking. Natural Prod. Rep. 27: 695-710; 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..
 Zinc: Deficiency common in plants and people

Barley grown with Zinc is deficient in 50% of the world’s agricultural soils
and without zinc and is recognized as the world’s most critical
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
Zn deficiency causes chlorosis, stunting, low yields
and death

Photo credits: South Dakota State University; IPNI; Howard F. Schwartz, Colorado State University, Bugwood.org; IRRI
 Ribosomal proteins represent the largest cellular
pool of Zn

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

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
 Zinc-fingers are found in many nucleic acid-binding
proteins

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

TFIIIA (the first iden9fied Zn-finger

protein) bound to DNA

Zn-fingers are parYcularly well

suited for interacYons 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.
 Manganese: Central to the oxygen-evolving reaction

The light-dependent Mn limitation interferes

reacYon that splits with O2 evolution
water and releases
oxygen depends on a

O2 released
Mn cluster

Mn

Symptoms of Mn deficiency

MnSOD is the
dominant SOD in
mitochondria

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; 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
Four photons are needed to charge the water-
splitting reaction

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
 In most cells molybdenum functions as Molybdenum
cofactor Moco
+Mo -Mo Mo is functional
when conjugated to
a pterin, as
Molybenum cofactor
(Moco) MoO 2- 4

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.
 MoO42- resembles SO42-, Mo and S transporters are
same family
MoO42-

MOT1, MOT2 Cytosol

Reversible
chelation MoO42-

CNX Moco
MoO42- resembles SO42- and pathway
high-affinity transport takes MOT2
place through MOT1 and
MOT2, members of the Moco MoO42-
Vacuole
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.
 A handful of plant enzymes use Mo

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

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.
 Nitrogenase, a bacterial enzyme, uses an Fe & Mo
cofactor, FeMoco

FeMoco is used by the bacterial

enzyme nitrogenase. This
enzyme is needed for symbiotic
nitrogen fixation that occurs in
N2-fixing plant nodules

FeMoco

N2 N2
Moco
Nitrogenase Note that FeMoco
NH4+
and Moco are
totally different
NH4+
structures

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.
 Nickel: Necessary but rarely limiting

The requirement for Ni was demonstrated not too long ago

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

Urease

% Germination
Ni in urease
active site Other Ni-dependent
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.
 Nickel transporters and chelators
Alyssum lesbiscum, a nickel
Ni uses many of the same transporters as Fe, and hyperaccumulator shows greatly
elevated levels of the amino acid
its toxicity may be due in part to competition with Fe
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.
 Toxic metals and metalloids

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 toxic to plants and humans and affects
millions

Arsenic is abundant, toxic

and a major human health
concern throughout south
and south-east Asia
where As naturally occurs
in soils and groundwaters

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.
 In severely arsenic-
affected areas, Eating As-
there are several contaminated
routes to human rice grain
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

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

As(III) As(OH)3

In rice, Lsi1 and Lsi2 transporters are

highly expressed for efficient Si uptake,
As(OH)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.
 Strategies to ameliorate As impacts
Increasing synthesis of
phytochelaYns, increased
sequestraYon 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) Phytochelatin
competing P As(III)
or Si to soil PO42-
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;
 Cadmium is an extremely toxic heavy metal
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
batteries
lung and kidney disease

JJ Harrison; Kanazawa Med

Molecular bases for Cd hyperaccumulation

Also CAX ca9on / proton exchangers

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
 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 (left) 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.
 Aluminium, a damaging element in acidic soils

Aluminum is the most abundant

metal in the Earth’s crust. At
neutral pH it is bound into
insoluble complexes. Acidic
Low pH Al3+
soils release Al3+ which arrests Soluble Aluminum
sensitive
root growth in sensitive plants
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.
 One important Al-tolerance strategy is organic acid
(OA) extrusion

Aluminum Aluminum
Al-tolerant varieYes excrete sensitive tolerant
much more organic acid than
sensiYve varieYes

Al3+
Al3+
Al3+ Al3+
Al3+
Al3+ Al3+
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.
 Al tolerance can be conferred by elevated
expression of a root cell malate transporter

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
 Mechanisms of aluminium tolerance include
exclusion & sequestration

Organic
acid
TranscripYonal secreYon
responses

Changes in
Vacuolar mitochondrial
sequestraYon metabolism

ROS accumulaYon
and detoxificaYon

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..
 Multiple mechanisms of Al tolerance 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

Ma, J., Chen, Z. and Shen, R. (2014). Molecular mechanisms of Al tolerance in gramineous plants. Plant Soil. 381: 1-12; see references therein
 Many plants naturally tolerate or accumulate
aluminium

Aluminium
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
and Al uptake is facilitated
Camellia sinensis

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 an essential micronutrient

Boron is found in Boron mine

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

Other uses include

•  Borosilicate glass
•  Laundry soap
•  Insecticide

1:5,000 Boric Acid 1:50,000 Boric Acid No Boric Acid

Warington, K. (1923). The effect of boric acid and borax on the broad bean and certain other plants. Ann. Bot. 37: 629-672
 Boron is indispensable for cell wall crosslinking

In plant cell walls, B crosslinks complex

Boron can crosslink
polysaccharides called Rhamnogalacturonan II (RG-II),
molecules by making diester made up of a backbone and four side chains
bridges between them
Rhamnogalacturonan II
Backbone
H3BO3 B(OH)4-
HO
HO − OH
B OH B
Two RG-II
HO
HO OH crosslinked
by boron
HO C C O − O C
− O
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. Complex Carbohydrate Research Center
 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
 Boron efflux mutants have developmental and cell
wall defects

RTE encodes BOR1, a boron efflux

transporter. Loss-of-function is thought to
prevent B from reaching the shoot in the
xylem transpiration stream
Boron Boron
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
 Natural variation in B transporter is associated with
B tolerance

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
 High B-tolerant wheat has been selected where soil
B is high

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.

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
 Boron transport is mediated in part by subcellular
localization of BOR1

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.
 Silicon is essential for some plants, beneficial for
many
Silicon (Si) is the Equisetum arvense In 1969 Chen and
second most abundant 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 SiO2 (insoluble
quartz sand), and in
biological systems as Diatoms form cell walls from silicon
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
 Silicon contributes to plant resistance to biotic &
abiotic stress
Powdery mildew on
control plant

Plant treated with

Si shows resistance

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

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. 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.
 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 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.
 Chlorine is an essential micronutrient

Chlorine deficiency Chlorine is an essential

micronutrient, although Cl
deficiency is not common

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
+ Cl - Cl
charged anion Malate

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.
 One of chloride’s main roles is to regulate cell turgor

Guard cells are one of the OPEN CLOSING

most intensely studied cells.
Stomatal opening occurs V-ATPase
PM-H+
when proton pumps energize Proton -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
 Other roles: O2-evolving complex and several special
metabolites

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

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 ; 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. Engvild, K.C. (1986). Chlorine-containing natural
compounds in higher plants. Phytochemistry. 25: 781-791; USDA
 Chloride transporters contribute to salinity tolerance
(salt exclusion)

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

Wheat grain Se Western Australia 0.001 mg / kg

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

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. 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
 Selenium can be found in several different forms in
plants

Selenate
Selenocysteine Selenocysteine can be
Selenate (SeO42-) is the major (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
sulfate SO42- transporters

Cystseine
Sulfate (Cys)
 Se metabolism and bioremediation involves S
assimilation genes
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

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.
 Summary and ongoing research

•  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
 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

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
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 to protect
people from the toxic
effects of arsenic and
cadmium
IRRI; CIAT; Water.org; Centre for Food Safety, Government of Hong Kong