© 2014 American Society of Plant Biologists

Plant Nutrition 1: Membrane

energetics and transport,

potassium nutrition and sodium

toxicity

© 2014 American Society of Plant Biologists

Outline

• Introduction to plant nutrition

• Overview of nutrient uptake and transport

• Energizing the membrane

• H+-ATPases and H+-PPases

• Potassium

• Uptake, transport and homeostasis

• Sodium

• Toxicity, transport and tolerance

© 2014 American Society of Plant Biologists

Plant nutrition: Introduction

44% Oxygen

42% Carbon

CO2,

photo-

synthesis

7% Hydrogen

H2O water

93% of plant

dry mass is

composed of

C, O and H

Plants are ~70 to

>90% water by

weight

7% Other,

from soil

K

Potassium

N

Nitrogen

P Phosphorus

Ca Calcium

Mg Magnesium

S Sulfur

Si Silicon

Cl Chlorine

Other

These elements are

obtained mainly from soil,

are often referred to as

mineral nutrients, and are

the subject of the topic

Plant Nutrition

© 2014 American Society of Plant Biologists

Plants assimilate mineral nutrients

from their surroundings

K+

K+

PO43-

PO43-

PO43-

NO3-

NO3-

K+ K+

K+

K+

K+

K+

PO43-

PO43-

PO43-

NO3-

NO3-

Nutrient assimilation is very

energetically demanding –

the nutrients have to be

moved against a

concentration gradient and

often a charge gradient

Algae

Bryophytes

Tracheophytes

© 2014 American Society of Plant Biologists

Nutrient uptake, assimilation and

utilization involve many processes Nutrient

acquisition

efficiency

Nutrient usage

efficiency

Root system

architecture

Root

exudates

Rhizosphere

microbiota

Symbioses

P

P

N N

NH3

Transporters

and pumps

Intercellular

transport

efficiency

X R-X

Assimilation and

remobilization

efficiency

Regulatory and

homeostatic

networks

© 2014 American Society of Plant Biologists

Nutrients removed from soils can be

replenished with fertilizers

Total nutrient requirement

Typical fertilizer application

Corn

Soy W

he

at

Co

tto

n

Ric

e

Kg

/ha

K

g/h

a

1000

800

600

400

200

0

0

200

400

Nitrogen

Phosphate

Potash

Magnesium

Sulfur

Most fertilizers

contain nitrogen

(N), phosphorus

(P) and potassium

(K). Some include

other elements

Fertilizers can be

complex waste

products or

refined blends of

nutrient salts

Plants remove nutrients from the soil

Source: USGS

© 2014 American Society of Plant Biologists

Global mineral nutrient resources

are unevenly distributed

Supply > Demand

Supply < Demand

FAO (2011) Current world fertilizer trends and outlook to 2015.

N

P2O5

K2O

© 2014 American Society of Plant Biologists

The global trade in fertilizers is

worth billions of dollars annually

IFIA

Ammonium Urea Potash Diammonium

phosphate

Monoammonium

phosphate

Phosphate

rock

Sulfur Sulfuric

acid

© 2014 American Society of Plant Biologists

How much is the right amount of

fertilizer to apply to a field?

Photo by Michael Russelle.

Species / variety

of plant: Different

plants have

different needs

Soil

characteristics:

Residual nutrients,

rate of nutrient

leaching, pH,

particle size,

presence of

microbes etc. affect

optimal application

Cultivation

practices: Is

unharvested

material removed,

or left to replenish

the soil?

Abiotic and biotic

factors: Temperature,

rain, stress and pests

or pathogens affect

nutrient needs

Developmental stage affects plant needs

Interactions between nutrients:

There are both positive and negative

interactions between various nutrients

Financial considerations:

Balancing the cost of fertilizers with

the gain reaped from their use

© 2014 American Society of Plant Biologists

Fertilizer use can cause

environmental and health problems Nitrogen fixation is

energy demanding

Phosphate and potash

mining is destructive

Image source: Lamiot; Alexandra Pugachevsky

Transport requires energy

Human and animal waste

can spread disease N O N

Nitrous oxide (N2O)

derived from fertilizer is a

major greenhouse gas

Nutrient runoff pollutes

waterways and can lead

to eutrophication

Plants need

nutrients, but their

application isn’t

always optimal or

sustainable – how

can plant science

contribute to

better practices?

© 2014 American Society of Plant Biologists

Pumps, channels

and carriers are the

molecular

mediators of these

processes

Membrane transport can

consume 1/3 of a cell’s

metabolic energy (or more)

Nutrients must be

transported across

membranes to enter

the plant

Cross membrane

into living cell in

root hair Symplastic or

transcellular pathway

Apoplastic pathway

Cross membrane

into living cell in

at endodermis

Bidirectional transport

between xylem parenchyma

cells and apoplastic

transpiration stream

Casparian strip

Nutrient uptake and transport:

Overview

© 2014 American Society of Plant Biologists

Plants assimilate mineral nutrients

mainly as cations or anions

μmol / g (dry wt)

Element Assimilated form

250 Potassium (K) K+

1000 Nitrogen (N) NO3-, NH4

+

60 Phosphorus (P)

HPO42-,

H2PO4-

30 Sulfur (S) SO42-

80 Magnesium (Mg)

Mg2+

125 Calcium (Ca) Ca2+

μmol / g (dry wt)

Element Assimilated form

2 Iron (Fe) Fe3+, Fe2+

0.002 Nickel (Ni) Ni2+

1 Manganese (Mn)

Mn2+

0.1 Copper (Cu) Cu2+

0.001 Molybdenum (Mo)

MoO42+

2 Boron (B) H3BO3

3 Chlorine (Cl) Cl-

0.3 Zinc (Zn) Zn2+

MACRONUTRIENTS MICRONUTRIENTS

See Taiz, L. and Zeiger, E. (2010) Plant Physiology. Sinauer Associates; Marschner, P. (2012) Mineral Nutrition of Higher Plants. Academic Press, London

© 2014 American Society of Plant Biologists

Nutrients are concentrated in the

plant relative to the environment

[K+]o

0.1 – 1 mM

[H2PO4-]o

[HPO42-]o

< 1 μM

[NO3-]o

<100 μM –

>1 mM

[NH4+]o

<100 μM –

>1 mM

[K+]i 50 - 100 mM

Soil abundance

(ranges or typical

values)

Cell [H2PO4

-]i [HPO4

2-]i

5 - 10 mM

[NO3-]i

10 mM

[NH4+]i

~1 mM

Energy is expended to assimilate nutrients

against a steep concentration gradient The driving force of

the nutrient’s

chemical gradient is

outwards

© 2014 American Society of Plant Biologists

Transport can be down or against an

electrochemical gradient Down an electrochemical gradient

(Diffusion or facilitated diffusion)

Through

membrane

Through

channel

Through

carrier

Against an electrochemical gradient

(Active transport)

ATP ADP + Pi

Primary active transport:

Directly coupled to ATP

hydrolysis

Secondary active

transport: Indirectly

coupled to ATP

hydrolysis

Symport Antiport

OUT

IN

© 2014 American Society of Plant Biologists

Solutes cross membranes through

different types of transporters

X

X

Pumps are often drawn

like lollipops, with a large

cytoplasmic catalytic

domain

Pumps: • Move solutes against a

chemical or charge gradient

• Couple transport to hydrolysis

of ATP or pyrophosphate

ATP ADP

ATP

ADP

The multisubunit vacuolar

proton pump VH+-ATPase

Reprinted from Schumacher, K. and Krebs, M. (2010). The V-ATPase: small cargo, large effects. Curr. Opin. Plant Biol. 13: 724-730 with permission from Elsevier.

© 2014 American Society of Plant Biologists

Solutes cross membranes through

different types of transporters

X

X X

Channels are often

drawn as two adjacent

ovals (or a cross-section

of a doughnut)

Channels: • are protein-formed holes in the membrane

• can be open or closed

• move one type of solute at a time

• do not provide an energy source for the

movement; solutes can only move down

their electrochemical gradient

Reprinted from Long, S.B., Campbell, E.B. and MacKinnon, R. (2005). Crystal structure of a mammalian voltage-dependent Shaker family K+ channel. Science. 309: 897-903 with permission from AAAS.

Side (L) and Top (R) views

of a potassium channel

© 2014 American Society of Plant Biologists

Solutes cross membranes through

different types of transporters

Carriers /

Coupled Transporters • are membrane proteins

• can be active or inactive

• can move more than one solute at a time

• The driver (usually H+ in plants) moves down

its electrochemical gradient, which provides

the energy for the co-transported solute’s

transport

X

X

Coupled transporters

are often drawn as

circles with arrows

indicating the direction

of flow for each ion

H+ H+

X

H+

Schematic domain structure

(L) and Top-down (R) views

of an HKT1 Na+ transporter

Cotsaftis, O., Plett, D., Shirley, N., Tester, M. and Hrmova, M. (2012). A two-staged model of Na+ exclusion in rice explained by 3D modeling of HKT transporters and alternative splicing. PLoS ONE. 7: e39865. Chérel,

I., Lefoulon, C., Boeglin, M. and Sentenac, H. (2014). Molecular mechanisms involved in plant adaptation to low K+ availability. J. Exp. Bot. 65: 833-848, by permission of Oxford University Press.

© 2014 American Society of Plant Biologists

Pumps, channels and carriers are

also involved in nutrient distribution

Reprinted from Ahmad, I. and Maathuis, F.J.M. (2014). Cellular and tissue distribution of potassium: Physiological relevance, mechanisms and regulation. J. Plant Physiol. 171: 708–714 with permission from Elsevier.

• Nutrient uptake is

just the first step

• The assimilated

nutrients have to be

transported to

where they are

needed, including

leaves and seeds

• The vacuole is an

important storage

compartment

© 2014 American Society of Plant Biologists

Cells expend ATP to maintain high

internal [K+] and exclude Na+

3Na+

2K+ 3Na+

2K+

In animal cells, a Na+ / K+

ATPase pumps both ions

In plant cells, Na+ and K+ transport is

indirectly coupled to ATP hydrolysis. The

plasma-membrane proton pump is the major

ATPase; it generates protonmotive force that

drives plant membrane transport

[K+]o

0.1 – 1 mM

[K+]i 50 - 100 mM

[Na+]o

50 - 100 mM

[Na+]i 10 mM

OUT IN

ATP ADP + Pi

H+ K+

ATPase

Δψ = -200 mV ΔpH

pH5.5 (acidic)

pH7.5 (alkaline)

PMF (protonmotive force) = Δψ + ΔpH

++++++++

- - - - - - -

Haruta, M. and Sussman, M.R. (2012). The effect of a genetically reduced plasma membrane protonmotive force on vegetative growth of Arabidopsis. Plant Physiol. 158: 1158-1171.

© 2014 American Society of Plant Biologists

The membrane as a battery:

Mitchell’s big idea

Mitchell was awarded the Nobel

Prize in Chemistry in 1978

Energy used to

form gradient Energy released

by dissipating

gradient; this

energy can

perform work

Peter Mitchell recognized the

importance of membrane-

bound ATPases in

membrane energetics. In this

drawing he highlights the

reversibility of the ATPase

Reprinted from Mitchell, P. (1961). Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism. Nature 191: 144-148.

© 2014 American Society of Plant Biologists

Plant cells have a membrane

potential (Em) -100 to -200 mV

0 mV

+100 mV -100 mV

Reference

electrode Recording

electrode

+

+ +

+

+ - - - -

Voltmeter

The electrical gradient is

maintained by the outward

pumping of protons (H+),

and the accumulation of

anions such as malate in

the cytosol

H+

Malate

© 2014 American Society of Plant Biologists

The electrochemical gradient is

important for ion transport

[K+]o

0.1 – 1 mM

[H2PO4-]o

[HPO42-]o

< 1 μM

[NO3-]o

<100 μM –

>1 mM

[NH4+]o

<100 μM –

>1 mM

[K+]i 50 - 100 mM

[H2PO4-]i

[HPO42-]i

5 - 10 mM

[NO3-]i

10 mM

[NH4+]i

~1 mM

The cell’s electrical gradient

drives anions OUT and cations IN

The electrochemical gradient

defines the energetic demands for

transport, and integrates the

electrical and concentration

gradients

Em = ~ -150 mV

© 2014 American Society of Plant Biologists

Nutrient transport requires energy

and selective transporters

Plant cells pump protons

out to make proton and

charge gradients - -

-

+

+

+

K+

K+

H+

Proton (H+) gradient

Charge gradient

The electrical (charge) and

proton gradients drive the

movements of ions across

membranes through selective

transporters

© 2014 American Society of Plant Biologists

Energizing the membrane: Plant H+-

ATPases and H+-PPases

Sze, H., Li, X. and Palmgren, M.G. (1999). Energization of plant cell membranes by H+-pumping ATPases: Regulation and biosynthesis. Plant Cell. 11: 677-689.

The plasma-membrane

H+-ATPase uses energy

from ATP to pump

protons out of the cell

The vacuolar-type proton

pumps transport protons into

the lumen of endomembrane

compartments (e.g., vacuole)

The H+-PPase uses energy

stored in pyrophosphate

The VH+-ATPase

is a multimeric

protein complex

© 2014 American Society of Plant Biologists

The PM H+-ATPase is a “master

enzyme” and “powerhouse”

See Palmgren, M.G. (2001). Plant plasma membrane H+-ATPases: Powerhouses for nutrient uptake. Annu. Rev. Plant Physiol. Plant Mol.

Biol. 52: 817-845; Figure adapted from Michelet, B. and Boutry, M. (1995). The plasma membrane H+-ATPase. Plant Physiol. 108: 1-6.

ADP H+ ATP

pH ~ 5 - 6

pH ~ 7.5 - - - - -

~ -150 mV

+ + + + +

By pumping protons out

of the cell, PM H+-

ATPases produce electric

and pH gradients

Cations Anions

Channels Antiporters

H+ H+

Symporters Uniporters The electrochemical

gradient produced by the

PM H+-ATPase drives other

transport processes

© 2014 American Society of Plant Biologists

PM H+-ATPases were first

characterized in fungi

Goffeau, A. and Slayman, C.W. (1981). The proton-translocating ATPase of the fungal plasma membrane. Biochim. Biophys. Acta (BBA) 639: 197-223 by permission of Elsevier; Serrano, R., Kielland-Brandt, M.C. and

Fink, G.R. (1986). Yeast plasma membrane ATPase is essential for growth and has homology with (Na+ + K+), K+- and Ca2+-ATPases. Nature. 319: 689-693; Hager, K.M., Mandala, S.M., Davenport, J.W., Speicher,

D.W., Benz, E.J. and Slayman, C.W. (1986). Amino acid sequence of the plasma membrane ATPase of Neurospora crassa: deduction from genomic and cDNA sequences. Proc. Natl. Acad. Sci. USA. 83: 7693-7697.

Predicted structure of cloned plasma-

membrane ATPase PMA1 from yeast. White bars indicate regions of homology with

other cation pumps

PM H+-ATPases were

initially cloned from fungi

Detailed physiological studies in

Neurospora characterized the

plasma membrane proton-

translocating ATPase

© 2014 American Society of Plant Biologists

Plant and fungal PM H+-ATPases are

members of a larger family

Reprinted by permission from Macmillan Publishers Ltd from Kühlbrandt, W. (2004). Biology, structure and mechanism of P-type ATPases. Nat. Rev. Mol. Cell Biol. 5: 282-295; see also Baxter, I., Tchieu, J., Sussman,

M.R., Boutry, M., Palmgren, M.G., Gribskov, M., Harper, J.F. and Axelsen, K.B. (2003). Genomic Comparison of P-Type ATPase Ion Pumps in Arabidopsis and Rice. Plant Physiol. 132: 618-628.

Mammalian

Na+/ K+-ATPase

Plant and

fungal plasma

membrane

H+-ATPases

3Na+

2K+ 3Na+

2K+

ATP ADP + Pi

H+

© 2014 American Society of Plant Biologists

Several differentially expressed

genes encode plant PM H+-ATPases

Arango, M., Gévaudant, F., Oufattole, M. and Boutry, M. (2003). The plasma membrane proton pump ATPase: the significance of gene subfamilies. Planta. 216: 355-365.Okumura, M., Inoue, S.-i.,

Takahashi, K., Ishizaki, K., Kohchi, T. and Kinoshita, T. (2012). Characterization of the plasma membrane H+-ATPase in the liverwort Marchantia polymorpha. Plant Physiol. 159: 826-834. DeWitt, N.D.

and Sussman, M.R. (1995). Immunocytological localization of an epitope-tagged plasma membrane proton pump (H+-ATPase) in phloem companion cells. Plant Cell. 7: 2053-2067. See also Okumura, M.,

Takahashi, K., Inoue, S.-i. and Kinoshita, T. (2012). Evolutionary appearance of the plasma membrane H+-ATPase containing a penultimate threonine in the bryophyte. Plant Signal. Behav. 7: 979 - 982.

AHA3 is highly expressed in phloem companion cells

Negative

control

Antibody

stain

Phylogeny showing functional

genes in Arabidopsis (AHA)

and four in the liverwort

Marcantia polymorpha (MpHA)

O

Other PM H+-ATPase-encoding genes are

expressed in other tissues, and many are

upregulated by stress or other factors

© 2014 American Society of Plant Biologists

PM H+-ATPase activity also is

regulated post-transcriptionally

The monomer is a

single polypeptide

with 10 membrane-

spanning domains

Reprinted from Kühlbrandt, W., Zeelen, J. and Dietrich, J. (2002). Structure, mechanism, and regulation of the Neurospora plasma membrane H+-ATPase. Science. 297: 1692-1696 with permission from AAAS;

Ottmann, C., et al. (2007). Structure of a 14-3-3 coordinated hexamer of the plant plasma membrane H+-ATPase by combining X-Ray crystallography and electron cryomicroscopy. Mol. Cell. 25: 427-440.

IN

OUT

ATP ADP + Pi

H+

Post-translational regulation

includes phosphorylation,

interaction with 14-3-3 proteins

and multimer formation

© 2014 American Society of Plant Biologists

Plant PM H+-ATPases are essential

for nutrient uptake and allocation

Sondergaard, T.E., Schulz, A. and Palmgren, M.G. (2004). Energization of transport processes in plants. Roles of the plasma membrane H+-ATPase. Plant Physiol. 136: 2475-2482.

Through their combined

actions, PM H+-ATPases

contribute to the movement of

nutrients throughout the plant

© 2014 American Society of Plant Biologists

PM H+-ATPases have diverse

physiological roles

• Loss of function of the PM-H+-ATPase is lethal

• PM H+-ATPases are required for

• nutrient assimilation and transport,

• cell growth, turgor and movement

• guard cell dynamics

Sondergaard, T.E., Schulz, A. and Palmgren, M.G. (2004). Energization of transport processes in plants. Roles of the plasma membrane H+-ATPase. Plant Physiol. 136: 2475-2482.

© 2014 American Society of Plant Biologists

Vacuolar pumps pump protons into

the vacuole and endocompartments

pH 3 - 6

pH 5 - 6

pH 7.5

Sze, H., Li, X. and Palmgren, M.G. (1999). Energization of plant cell membranes by H+-pumping ATPases: Regulation and biosynthesis. Plant Cell. 11: 677-689.

Isayenkov, S., Isner, J.C. and Maathuis, F.J.M. (2010). Vacuolar ion channels: Roles in plant nutrition and signalling. FEBS letters. 584: 1982-1988.

Em = ~ -30 mV

Protons are pumped into the

vacuole by:

• Vacuolar H+-ATPases (VH+-

ATPases) and

• Vacuolar pyrophosphatases

(H+-PPases)

H+

H+

H+

H+

PPi 2 x Pi

ADP + Pi H+

ATP

© 2014 American Society of Plant Biologists

The VH+-ATPase is a large multi-

subunit enzyme

Schumacher, K. and Krebs, M. (2010). The V-ATPase: small cargo, large effects. Curr. Opin. Plant Biol. 13: 724-730; Reprinted by permission from Macmillan Publishers Ltd from Nishi, T. and Forgac, M. (2002). The vacuolar (H+)-

ATPases — nature's most versatile proton pumps. Nat. Rev. Mol. Cell Biol. 3: 94-103; Ward, J.M. and Sze, H. (1992). Subunit composition and organization of the vacuolar H+-ATPase from oat roots. Plant Physiol. 99: 170-179.

Hydrolyzes ATP to

generate proton gradient Uses proton gradient

to synthesize ATP

VH+-ATPase F-ATPase

VH+-ATPases are related to mitochondrial F-ATPases,

even though their transport directions are reversed

The knob-like V1

domains of VH+-ATPase

are visible on a plant

membrane

© 2014 American Society of Plant Biologists

VH+-ATPases contribute to growth,

salt tolerance & ion uptake / storage

Gogarten, J.P., Fichmann, J., Braun, Y., Morgan, L., Styles, P., Taiz, S.L., DeLapp, K. and Taiz, L. (1992). The use of antisense mRNA to inhibit the tonoplast H+ ATPase

in carrot. Plant Cell. 4: 851-864. Krebs, M., Beyhl, D., Görlich, E., Al-Rasheid, K.A.S., Marten, I., Stierhof, Y.-D., Hedrich, R. and Schumacher, K. (2010). Arabidopsis V-

ATPase activity at the tonoplast is required for efficient nutrient storage but not for sodium accumulation. Proc. Natl. Acad. Sci. USA. 107: 3251-3256.

Phenotypes associated with

decreased VH+-ATPase activity:

• Decreased growth rate

• Male sterility

• Altered nutrient storage

capabilities

Decreased growth rate in carrot

plants expressing an antisense

VH+-ATPase A construct

A mutant lacking functional tonoplast

VH+-ATPase is sensitive to Zn2+ toxicity

(cannot sequester it into vacuole)

© 2014 American Society of Plant Biologists

The VH+-ATPases have different

roles in different compartments

VHA-a2 localizes to the

vacuolar membrane

VHA-a1 localizes to the

trans-Golgi network

Mutants lacking one or the other

isoform reveal that the

VH+-ATPases have different roles

in different compartments

In Arabidopsis, three

genes encode VHA-a,

and their gene products

localize to different

subcellular compartments

TEM showing VHA-a1(black

dots) localized in TGN

Nishi, T. and Forgac, M. (2002). The vacuolar (H+)-ATPases — nature's most versatile proton pumps. Nat. Rev. Mol. Cell Biol. 3: 94-103. Dettmer, J., Hong-Hermesdorf,

A., Stierhof, Y.-D. and Schumacher, K. (2006). Vacuolar H+-ATPase activity is required for endocytic and secretory trafficking in Arabidopsis. Plant Cell. 18: 715-730.

© 2014 American Society of Plant Biologists

VH+-ATPases have multiple

functions in plants and animals VH+-ATPases have roles in

pH homeostasis in

endomembranes and also

in membrane trafficking

Functions in plants

• Development

• Cell expansion

• Nutrient assimilation

Functions in humans

• Development

• Kidney function

• Bone resorption

• Tumor cell metastasis

Reprinted from Schumacher, K. and Krebs, M. (2010). The V-ATPase: small cargo, large effects. Curr. Opin. Plant Biol. 13: 724-730 with permission from Elsevier.

© 2014 American Society of Plant Biologists

The vacuolar pyrophosphatase (H+-

PPase) uses PPi as energy source

Madprime

H+

PPi 2 x Pi

H+-PPase uses PPi

as an energy source

to pump protons into

the vacuole

© 2014 American Society of Plant Biologists

Plants have 2 types of H+-PPases

Reprinted from Drozdowicz, Y.M. and Rea, P.A. (2001). Vacuolar H+ pyrophosphatases: from the evolutionary backwaters into the mainstream. Trends Plant Sci. 6: 206-211 with permission from Elsevier; Gaxiola, R.A.,

Sanchez, C.A., Paez-Valencia, J., Ayre, B.G. and Elser, J.J. (2012). Genetic manipulation of a “vacuolar” H+-PPase: From salt tolerance to yield enhancement under phosphorus-deficient soils. Plant Physiol. 159: 3-11.

Blue indicates

eubacteria

Red indicates

Archaea

Green indicates

eukaryotes

Eukaryotes with H+-

PPases are limited to

plants and green

algae (circled in

green) and parasitic

protists (circled in

orange)

Type 2:

Golgi-localized

K+ insensitive

Strongly inhibited by Ca2+

Type 1:

Tonoplast-localized

Require K+ for activity

Moderately inhibited by Ca2+

© 2014 American Society of Plant Biologists

Each H+-PPase subunit has 16

membrane spanning domains

PPi

Reprinted by permission from Macmillan Publishers Ltd from Lin, S.-M., Tsai, J.-Y., Hsiao, C.-D., Huang, Y.-T., Chiu, C.-L., Liu, M.-H., Tung, J.-Y., Liu,

T.-H., Pan, R.-L. and Sun, Y.-J. (2012). Crystal structure of a membrane-embedded H+-translocating pyrophosphatase. Nature. 484: 399-403.

H+-PPase functions

as a homodimer

PPi

Structure of a plant H+-PPase

© 2014 American Society of Plant Biologists

H+-PPases have many physiological

roles

Gaxiola, R.A., Li, J., Undurraga, S., Dang, L.M., Allen, G.J., Alper, S.L. and Fink, G.R. (2001). Drought- and salt-tolerant plants result from overexpression of the AVP1 H+-pump. Proc. Natl, Acad. Sci. USA. 98:

11444-11449. Yang, H., Zhang, X., Gaxiola, R.A., Xu, G., Peer, W.A. and Murphy, A.S. (2014). Over-expression of the Arabidopsis proton-pyrophosphatase AVP1 enhances transplant survival, root mass, and fruit

development under limiting phosphorus conditions. J. Exp. Bot. 65: 3045-3053 by permission of Oxford University Press.

Plants overexpressing H+-PPase

show enhanced drought tolerance

Altering H+-PPase expression affects:

Salinity and drought tolerance

Nutrient uptake

Auxin transport

Phosphate uptake

Fruit ripening …….

Accelerated fruit ripening in tomato

plants overexpressing H+-PPase

© 2014 American Society of Plant Biologists

Energizing the membrane: Summary

• Electrochemical gradients across membranes

(pH and charge) store energy that can be used

to move materials across the membrane

• Plant cells are energized by proton pumps that

reside in the plasma membrane or

endomembranes

© 2014 American Society of Plant Biologists

K+ and Na+ - “The twins”. So alike

yet so different

K

Sodium (Na) and potassium (K):

• Same column of the periodic table

• Both have a single electron in the

outer shell so form monovalent

cations

• Both are very abundant elements

Potassium

deficiency

NaCl

toxicity

And yet, potassium is an

essential nutrient, and

sodium frequently is toxic

Benito, B., Haro, R., Amtmann, A., Cuin, T.A. and Dreyer, I. (2014).The twins K+ and Na+ in plants. J. Plant Physiol. 171: 723–731. FAO

© 2014 American Society of Plant Biologists

Potassium uptake, transport and

homeostasis

Regulates

stomatal

conductance,

photosynthesis

and transpiration

Maintains turgor

and reduces wilting

Strengthens

cell walls Maintains ionic

homeostasis Stimulates

photosynthate

translocation

Enhances

fertility

Promotes stress

tolerance

See Wang, M., Zheng, Q., Shen, Q. and Guo, S. (2013). The critical role of potassium in plant stress response. Intl. J. Mol. Sci. 14: 7370-7390; Sin Chee Tham /Photo; Purdue extension; Onsemeliot.

Symptoms of

potassium deficiency

[K+] in soil = ~0.1 – 1 mM

[K+] in plant cell

cytoplasm = ~100 mM

Potassium is an essential macronutrient

Regulates

enzyme activities

© 2014 American Society of Plant Biologists

Potassium fertilizers are mined from

underground reserves as “potash”

Almost half of the world’s reserved of potash

are found in Saskatchewan, Canada

Potash is a term that encompasses

many forms of potassium:

• KCl (potassium chloride, aka sylvite)

• K2SO4 (potassium sulfate)

• K2CO3 (potassium carbonate)

• K2Ca2Mg(SO4)4·2H2O (polyhalite)

• etc.

Canada Potash; Lmbuga

KCl, sylvite

For historical reasons, potash

is measured in units of K2O

equivalents, even though it is

rarely found in the form of K2O

© 2014 American Society of Plant Biologists

Potash provides K+ for fertilizers,

which supplement natural sources

manure

decomposition

Terrestrial

cycle: Plant /

Animal / Soil Underground reserves

Water with

dissolved K+

salts returned

to surface

Water

pumped

underground

Salts

recovered by

evaporation

90 – 98%

insoluble

minerals

1 – 3%

exchangeable

salts

0.1 – 0.2% soil

solution K+

Potash

fertilizer

application

Adapted from International Potash Institute

© 2014 American Society of Plant Biologists

Potash prices can be volatile and

there are few suppliers

1.06 cm

Canada is #1

in production

(11.2 Mt) and

reserves

(4,400 Mt)

Russia is #2

in production

(7.4 Mt) and

reserves

(3,300 Mt)

Brazil

3.2 Mt

210 Mt

Chile

0.8 Mt

130 Mt

US

1.1 Mt

130 Mt

China

3.2 Mt

210 Mt

Belarus

5.5 Mt

750 Mt

World

reserves

9500 Mt

World

production

(2011)

37 Mt

Jordan

1.4 Mt

40 Mt

Israel

2.0 Mt

40 Mt

Germany

3.3 Mt

150 Mt

Spain

0.4 Mt

20 Mt

UK

0.4 Mt

22 Mt

Adapted from International Potash Institute

© 2014 American Society of Plant Biologists

Potassium is an essential plant

nutrient

Reprinted from Maathuis, F.J.M. (2009). Physiological functions of mineral macronutrients. Curr. Opin. Plant Biol. 12: 250-258 with permission from Elsevier.

K+ uptake

involves high

and low affinity

transporters

K+ is a counter ion for

negatively charged molecules

including DNA and proteins

K+ is a cofactor for

some enzymes

As the major cation in

the vacuole, K+

contributes to cell

expansion and

movement, including

that of guard cells

K+ moves in and

out of the vacuole

through specific

transporters

© 2014 American Society of Plant Biologists

Early studies of potassium uptake in

plants: Biphasic uptake

Epstein, E., Rains, D.W., and Elzam, O.E. (1963). Resolution of dual mechanisms of potassium absorption by barley roots. Proc. Natl. Acad. Sci. USA. 49: 684 – 692;

Gierth, M. and Mäser, P. (2007). Potassium transporters in plants – Involvement in K+ acquisition, redistribution and homeostasis. FEBS Lett. 581: 2348-2356.

KCl (mM)

Low affinity

transport

High affinity

transport

Epstein et al showed

two phases of K+

uptake in barley roots

More recently Arabidopsis mutants

suggest HAK5 and AKT1 mediate high

and low affinity uptake, respectively.

These data are their contributions

inferred from mutant studies.

© 2014 American Society of Plant Biologists

More energy must be expended to

take up K+ when it is scarce

See Britto, D.T. and Kronzucker, H.J. (2008). Cellular mechanisms of potassium transport in plants. Physiol. Plant. 133: 637-650; Nieves-Cordones, M., Alemán, F.,

Martínez, V. and Rubio, F. (2014). K+ uptake in plant roots. The systems involved, their regulation and parallels in other organisms. J. Plant Physiol. 171: 688–695.

K+ uptake from low [K+]ext

requires more energy than

when [K+]ext is higher

K+ K+ H+

H+

ATP

2 x H+

2 x ATP

At very high [K+]ext, non-

selective cation channels

(NSCC) also may contribute

Low affinity

transport

High affinity

transport

© 2014 American Society of Plant Biologists

There are several types of coupled

transporters for K+

Chérel, I., Lefoulon, C., Boeglin, M. and Sentenac, H. (2014). Molecular mechanisms involved in plant adaptation to low K+ availability. J. Exp. Bot. 65: 833-848, by permission of Oxford

University Press; Gierth, M. and Mäser, P. (2007). Potassium transporters in plants – Involvement in K+ acquisition, redistribution and homeostasis. FEBS letters. 581: 2348-2356.

c

c KT/KUP/HAK transporters are

responsible for much of the high-affinity

uptake into roots. There are 13 genes

in Arabidopsis and 27 in rice

K+ H+ They are K+ / H+

symporters

Some members of the large CPA (Cation Proton

Antiporter) family contribute to K+ uptake

Blue indicates

preferential K+

uptake

K+ / H+ antiporters

© 2014 American Society of Plant Biologists

There are 15 genes encoding

potassium channels in Arabidopsis

Used with permission from Hedrich, R. (2012). Ion channels in plants. Physiol.Rev. 92: 1777-1811.

9 Shaker-

type (Kv)

(Plasma

membrane)

5 Two Pore K+

(TPK) channels

(mostly tonoplast)

1 Non-selective

two-pore cation

(TPC) channel

1 Kir-like

potassium

channel

© 2014 American Society of Plant Biologists

Shaker channels form as tetramers

that can be heteromers

Hedrich, R. (2012). Ion channels in plants. Physiol.Rev. 92: 1777-1811; Chérel, I., Lefoulon, C., Boeglin, M. and Sentenac, H. (2014). Molecular mechanisms involved in plant adaptation

to low K+ availability. J. Exp. Bot. 65: 833-848, by permission of Oxford University Press; Reprinted from Anschütz, U., Becker, D. and Shabala, S. (2014), Going beyond nutrition:

Regulation of potassium homoeostasis as a common denominator of plant adaptive responses to environment. J. Plant Physiol. 171: 670-687 with permission from Elsevier.

Each monomer has

6 transmembrane

domains

Model of a Shaker K+ channel tetramer with

each monomer shown in a different color

© 2014 American Society of Plant Biologists

Ion flux through Kv channels is

voltage driven and voltage gated

K+ K+

Em < EK

When the channel is open,

current direction is

determined and driven by

membrane potential

Em > EK

IV curve showing current (y axis)

at different voltages (x axis)

Reprinted from Dreyer, I., and Blatt, M. (2009). What makes a gate? The ins and outs of Kv-like K+ channels in plants. Trends Plant Sci. 14: 383 – 390 with permission from Elsevier.

© 2014 American Society of Plant Biologists

Kv channels are voltage gated: Their

“openness” is voltage sensitive

Voltage-sensitive potassium

channels (Kv) are closed at

some membrane voltages

Inward rectifying channels

are open with a fixed

midpoint voltage V1/2

Notice that the relative

conductance is the relative

probability that the gate is open

V1/2, the half-maximal

activation voltage, is

the key parameter

Outward rectifying

channels are open with a

midpoint voltage that is

[K]out dependent

Reprinted from Dreyer, I., and Blatt, M. (2009). What makes a gate? The ins and outs of Kv-like K+ channels in plants. Trends Plant Sci. 14: 383 – 390 with permission from Elsevier.

© 2014 American Society of Plant Biologists

Current depends on the product of

membrane voltage and gating

Gating Current Voltage

potential x =

Inward

rectifying

channel

Outward

rectifying

channel

Reprinted from Dreyer, I., and Blatt, M. (2009). What makes a gate? The ins and outs of Kv-like K+ channels in plants. Trends Plant Sci. 14: 383 – 390 with permission from Elsevier.

© 2014 American Society of Plant Biologists

Different Shaker-type channels

respond differently to voltage

Although their primary

sequences are similar,

some are voltage-gated and

some not, and some are

inward rectifying and some

outward rectifying

Kout

Ksilent

Kweak

Kin

Reprinted from Dreyer, I., and Blatt, M. (2009). What makes a gate? The ins and outs of Kv-like K+ channels in plants. Trends Plant Sci. 14: 383 – 390 with permission from Elsevier;

Pilot, G., Pratelli, R., Gaymard, F., Meyer, Y. and Sentenac, H. (2003). Five-group distribution of the Shaker-like K+ channel family in higher plants. J. Mol. Evol. 56: 418-434.

© 2014 American Society of Plant Biologists

Example: SKOR is open only when

Em is > EK, so K+ only moves out

In the case of

SKOR, gating is

also sensitive to

external [K+]

Reprinted with permission from Gaymard, F., Pilot, G., Lacombe, B., Bouchez, D., Bruneau, D., Boucherez, J., Michaux-Ferrière, N., Thibaud, J.-B. and

Sentenac, H. (1998). Identification and disruption of a plant shaker-like outward channel involved in K+ release into the xylem sap. Cell. 94: 647-655.

Outer root

layer

Vascular

cylinder

Potassium efflux is

necessary for K+ to

enter the xylem

stream and move

from root to shoot

Depolarized

membrane (less

negative inside) so

K+ flows out

through open

SKOR channels

K+

K+

-

+

OUT

IN SKOR expression in

the cells surrounding

the root xylem

influx

efflux K+ K+

K+

© 2014 American Society of Plant Biologists

Guard cells are model systems for

the study of K+ transport OPEN CLOSING

H+

H+

H+

H+

H+

H+

K+ K+

KAT

PM-H+-ATPase

V-ATPase

V-PPase

GORK

K+

K+

A-

A-

TPK

TPC

Hills, A., Chen, Z.-H., Amtmann, A., Blatt, M.R. and Lew, V.L. (2012). OnGuard, a computational platform for quantitative kinetic modeling of guard cell physiology. Plant Physiol. 159: 1026-1042 Chen, Z.-H., Hills, A.,

Bätz, U., Amtmann, A., Lew, V.L. and Blatt, M.R. (2012). Systems dynamic modeling of the stomatal guard cell predicts emergent behaviors in transport, signaling, and volume control. Plant Physiology. 159: 1235-1251.

The depth and breadth of

information available for

stomatal guard cells has

made them the premier cell

system in plants for studies

of membrane transport,

signaling, and homeostasis

© 2014 American Society of Plant Biologists

The activity of membrane pumps

and channels is coordinated ABA

K+in

channels K+

out

channels

Anion

channels

H+-PPase V-H+-ATPase

PM-H+-ATPase

V-K+in

channels

V-K+out

channels

V-Anion

channels

[Ca2+]cyt Protein

phosphorylation

pHcyt

Stomata opening Stomata closing

Plasma

membrane

Tonoplast

Adapted from Chen, Z.H., and Blatt, M.R. (2010) Membrane Transport in Guard Cells. In: Encyclopedia of Life Sciences (ELS). John Wiley & Sons, Ltd: Chichester

© 2014 American Society of Plant Biologists

Potassium homeostasis: Responses

to low K+ availability

Adapted from Chérel, I., Lefoulon, C., Boeglin, M. and Sentenac, H. (2013). Molecular mechanisms

involved in plant adaptation to low K+ availability. J. Exp. Bot. 65: 833-848.

Low K

Membrane

hyperpolarization

More efficient

uptake through

K+ channels

Transcriptional

induction of HAK5

K+ channel

Hormonal changes

(auxin, ethylene)

Calcium

signaling

K+ uptake

Enhanced root

growth and

gravitropic

responses

Direct effects

Indirect effects

© 2014 American Society of Plant Biologists

K+ mobilization is critical for K+

homeostasis

Adapted from Amtmann, A., and Leigh, R. (2010). Ion homeostasis. In Abiotic Stress Adaptation in Plants: Physiological, Molecular and Genomic

Foundation, A. Pareek, S.K. Sopory, H.J. Bohnert and Govindjee (eds) (Dordrecht, The Netherlands: Springer), pp. 245 – 262.

Cytosol

Vac.

Supraoptimal K+

can be stored in

the vacuole As K+ becomes

limiting, it becomes

preferentially allocated

to the cytosol

© 2014 American Society of Plant Biologists

K+ mobilization is critical for K+

homeostasis

Cytosol

Vac.

Prioritized

Non-

Prioritized

As K+ becomes

limiting, it becomes

preferentially allocated

to the cytosol

K+ can be remobilized

from less essential tissues

into prioritized tissues

such as growing and

photosynthetic tissues

Adapted from Amtmann, A., and Leigh, R. (2010). Ion homeostasis. In Abiotic Stress Adaptation in Plants: Physiological, Molecular and Genomic

Foundation, A. Pareek, S.K. Sopory, H.J. Bohnert and Govindjee (eds) (Dordrecht, The Netherlands: Springer), pp. 245 – 262.

© 2014 American Society of Plant Biologists

Summary: Potassium uptake,

transport and homeostasis

• Potassium is an essential macronutrient

required in large amounts

• Potassium is transported by channels and

transporters which are regulated transcriptionally

and post-transcriptionally, by membrane voltage

potential, and signals such as pH, Ca2+ and

hormones

• K+ uptake, transport and remobilization are

regulated extensively to ensure that the plant’s

critical tissues are preferentially supported

© 2014 American Society of Plant Biologists

Sodium toxicity, transport and

tolerance

Colum, P. (1918). The Adventures of Odysseus and the Tale of Troy. Project Gutenberg; USDA, USDA, Peggy Greb; FAO

To demonstrate his (fake)

madness, Odysseus plowed

salt into his field

You can’t take salt

out of soil easily;

once it is there it

stays there

© 2014 American Society of Plant Biologists

Saline soils occur worldwide and are

becoming more abundant

FAO; From: Corbishley, J. and Pearce, D., Growing trees on salt-affected land. ACIAR Impact Assessment Series Report No. 51,

July 2007; See also Munns, R. and Tester, M. (2008). Mechanisms of salinity tolerance. Annu. Rev. Plant Biol. 59: 651-681..

Global distribution of salt-affected soils

Approximately 7 % of world’s

land area and 30 % of

irrigated land is salt affected

Area of salinization

© 2014 American Society of Plant Biologists

Coastal and inland soils become

saline for different reasons

European Soil Portal

Inland areas:

Low rainfall and high rates

of evotranspiration

Seawater

Ground

water

Soil

Sea spray

Seawater

Soil Rising sea

level Lowering

ground

water table

France

Spain

Coastal

Coastal areas: Saline soils

occur due to intrusion of

seawater aggravated by

storms, rising sea levels

and lowering water tables

© 2014 American Society of Plant Biologists

Melting land ice is raising sea levels

and threatening agricultural lands

Reprinted from Nicholls, R.J. and Cazenave, A. (2010). Sea-level rise and its impact on coastal zones. Science. 328: 1517-1520 by permission of AAAS; Vermeer, M. and Rahmstorf, S. (2009). Global

sea level linked to global temperature. Proc. Natl. Acad. Sci. 106: 21527-21532.See also Cazenave, A. and Llovel, W. (2010). Contemporary sea level rise. Annu. Rev. Marine Sci. 2: 145-173. IRRI

Low-lying countries such as Bangladesh are

particularly vulnerable –here a farmer

surveys the damage caused by a cyclone

Se

a leve

l ch

an

ge

(cm

)

Sea levels are

expected to be

>1 m higher by 2100

© 2014 American Society of Plant Biologists

The San Francisco Bay and Delta are

becoming increasingly salty

San Francisco,

California

Sacramento

River

San

Joaquin

River • As less water flows through rivers into the

delta and bay, salty water moves inland

• Decreased river flows are caused by drought

(less rain) and increased diversion of water to

other parts of the state

Increased

evaporation causes

river water also to be

more salty

DeltaModelingAssociates

© 2014 American Society of Plant Biologists

Inland, many soils lie above ancient

deep salt deposits that can move up

Salty water

Salty

water

Salty

water

Department of Agriculture and Rural Affairs (1980). ‘Managing Salinity: Ensuring a Farming Future’. The State of Victoria

Clearing native vegetation often leads to soil salinization

© 2014 American Society of Plant Biologists

Irrigation also contributes to soil

salinity by mobilizing deep salts

Without irrigation

rainwater does not

penetrate below the

rootzone

Excessive irrigation

penetrates into deeper,

salty soils, dissolves the

salts and draws them

upwards into the rootzone

Rain

Evaporation

Rain

Salt

(dissolved)

© 2014 American Society of Plant Biologists

How can we address the problems

caused by soil salinization?

Identify

responses to salt

stress in salt-

sensitive species

(glycophytes)

Introduce salinity-tolerance

traits into crop plants through

breeding and engineering

Identify halophytes

that can be used as

food or energy crops

Munns, R., James, R.A., Xu, B., Athman, A., Conn, S.J., Jordans, C.,

Byrt, C.S., Hare, R.A., Tyerman, S.D., Tester, M., Plett, D. and

Gilliham, M. (2012). Wheat grain yield on saline soils is improved by

an ancestral Na+ transporter gene. Nat Biotech. 30: 360-364.

CSIRO; The State of Victoria; Maurice Chédel; Marco Schmidt

Avoid adding to the

problem by better

management of

fragile soil systems

Areas of concern

Learn about salt

tolerance from

naturally salt-

tolerant species

(halophytes)

Arthrocnemum

macrostachyum

Study salt-tolerant relatives

of crop plants

Chenopodium

quinoa

Thinopyrum

ponticum

Salicornia europaea

Geng, Y., Wu, R., Wee, C.W., Xie, F., Wei, X., Chan, P.M.Y.,

Tham, C., Duan, L. and Dinneny, J.R. (2013). A spatio-temporal

understanding of growth regulation during the salt stress response

in Arabidopsis. Plant Cell. 25: 2132-2154.

© 2014 American Society of Plant Biologists

Plant species have a broad range of

salinity tolerances

Arabidopsis

and rice are

quite sensitive

Saltbush (Atriplex amnicola)

is a halophyte that can

tolerate very salty soil

Reprinted by permission of Annual Reviews from Munns, R. and Tester, M. (2008). Mechanisms of salinity tolerance. Annu. Rev. Plant Biol. 59: 651-681.

Q. Can we identify and

exploit the mechanistic

basis of increased

salinity tolerance?

A. YES!

© 2014 American Society of Plant Biologists

Mechanisms of sodium toxicity and

tolerance

Adapted from Horie, T., Karahara, I. and Katsuhara, M. (2012). Salinity tolerance mechanisms in glycophytes: An overview with the central focus on rice plants. Rice. 5: 11; see also Munns, R. and Tester, M. (2008). Mechanisms of salinity

tolerance. Annu. Rev. Plant Biol. 59: 651-681 and Shabala, S. and Pottosin, I. (2014). Regulation of potassium transport in plants under hostile conditions: implications for abiotic and biotic stress tolerance. Physiol. Plant. 151: 257-279.

SALINITY STRESS

Ionic stress:

K+ deficiency /

excess Na+ influx Inhibition of:

water uptake,

growth,

photosynthesis

Osmotic

adjustment:

Accumulation of

solutes

Inhibition of:

enzyme activity,

protein synthesis,

photosynthesis

Leaf senescence

Ion homeostasis:

Na+ extrusion,

Na+ exclusion,

Na+ compartmentation

Osmotic stress

Oxidative

stress

Detoxification

strategies

© 2014 American Society of Plant Biologists

General sodium tolerance strategy:

Keep sodium out of cytosol & shoot

IN

OUT

“OUT”

1. Keep Na+ from

entering plant / cells

2. Pump out any

Na+ that leaks in

3. Compartmentation

of Na+ in vacuole

4. Extrude Na+

via salt glands

6. Synthesize

compatible solutes for

osmotic balance

Na+

Na+

Na+

Na+

K+ 5. Accumulate K+

to maintain a high

ratio of K+ to Na+

Compatible

solutes

7. Prevent Na+

from moving into

the shoot and

leaves

© 2014 American Society of Plant Biologists

Na+ transport and exclusion is an

integral part of Na+ tolerance

As Na+ becomes more

prevalent, it is preferentially

sequestered into the

vacuole via transporters

Cytosol

Vac.

Na+ can be sequestered in

less essential tissues and

excluded from growing

and photosynthetic tissues

Prioritized

Non-

Prioritized

Adapted from Amtmann, A., and Leigh, R. (2010). Ion homeostasis. In Abiotic Stress Adaptation in Plants: Physiological, Molecular and Genomic

Foundation, A. Pareek, S.K. Sopory, H.J. Bohnert and Govindjee (eds) (Dordrecht, The Netherlands: Springer), pp. 245 – 262.

© 2014 American Society of Plant Biologists

Ion pumps, channels & carriers

contribute to Na+ tolerance

H+

H+

H+ ATP ADP + Pi

ATP ADP+ Pi

PP 2 x Pi

Na+

H+

Na+ H+

H+ Na+

SOS1

VH+-ATPase H+-PPase NHX

PM-H+-ATPase

SOS1, NHX8

NSCC Na+

Na+ HKT

See Maathuis, F.J.M. (2014). Sodium in plants: perception, signalling, and regulation of sodium fluxes. J. Exp. Bot. 65: 849-858.

© 2014 American Society of Plant Biologists

HKTs have essential roles in salt

exclusion and salinity tolerance

HKT stands for “high

affinity K+ transport” but

they also contribute to

Na+ transport

Na+ Root

Leaf Xylem

Expression of HKT1 in the cells

surrounding xylem in the root helps

prevent Na+ from reaching the

photosynthetic cells in the shoot

Adapted from Davenport, R.J., MuÑOz-Mayor, A., Jha, D., Essah, P.A., Rus, A.N.A. and Tester, M. (2007). The Na+ transporter AtHKT1;1 controls retrieval of Na+ from the xylem

in Arabidopsis. Plant Cell Environ. 30: 497-507. Reprinted by permission from Macmillan Publishers Ltd from Ren, Z.-H., Gao, J.-P., Li, L.-G., Cai, X.-L., Huang, W., Chao, D.-Y.,

Zhu, M.-Z., Wang, Z.-Y., Luan, S. and Lin, H.-X. (2005). A rice quantitative trait locus for salt tolerance encodes a sodium transporter. Nat Genet. 37: 1141-1146.

Expression pattern in rice root;

blue indicates gene expression

© 2014 American Society of Plant Biologists

HKT1 expression level and activity is

correlated with Na+-tolerance

Mäser, P., Eckelman, B., Vaidyanathan, R., Horie, T., Fairbairn, D.J., Kubo, M., Yamagami, M., Yamaguchi, K., Nishimura, M., Uozumi, N., Robertson, W., Sussman, M.R. and Schroeder,

J.I. (2002). Altered shoot/root Na+ distribution and bifurcating salt sensitivity in Arabidopsis by genetic disruption of the Na+ transporter AtHKT1. FEBS letters. 531: 157-161.

Salt-tolerance in varieties of

rice, wheat and barley has

been genetically mapped to

variation in HKT activity

Loss of function =

more salt sensitive

Gain of function =

more salt tolerant

© 2014 American Society of Plant Biologists

Monocots have two types of HKTs

with different functions

Subfamily 1 Subfamily 2

Reprinted from Véry, A.-A., Nieves-Cordones, M., Daly, M., Khan, I., Fizames, C. and Sentenac, H. (2014). Molecular biology of K+ transport across the plant cell membrane: What do we learn from comparison

between plant species? J. Plant Physiol. 171: 748-769 with permission from Elsevier. See also Horie, T., Costa, A., Kim, T.H., Han, M.J., Horie, R., Leung, H.-Y., Miyao, A., Hirochika, H., An, G. and Schroeder, J.I.

(2007). Rice OsHKT2;1 transporter mediates large Na+ influx component into K+-starved roots for growth. EMBO J. 26: 3003-3014.

Type 1

Retrieval of Na+ from

transpiration stream

Found in all plants

Type 2

Postulated role in nutritional

Na+ uptake, when [K+] very low

(activity suppressed by K+)

Only present in monocots

Root

Na+

K+

Na+ Root

Leaf Xylem

© 2014 American Society of Plant Biologists

NHX (Sodium / proton exchangers)

are part of the CPA family

Chérel, I., Lefoulon, C., Boeglin, M. and Sentenac, H. (2013). Molecular mechanisms involved in plant adaptation to low K+ availability. J. Exp. Bot. 65: 833-848. Gierth, M. and Mäser, P.

(2007). Potassium transporters in plants – Involvement in K+ acquisition, redistribution and homeostasis. FEBS letters. 581: 2348-2356. Chanroj, S., Wang, G., Venema, K., Zhang, M.W.,

Dalwiche, C.F., and Sze, H. (2012). Conserved and diversified gene families of monovalent cation / H+ antiporters from algae to flowering plants. Front. Plant Sci. 3: 25.

Arabidopsis: 8 NHX transporters

AtNHX 1 – 4 Vacuole

AtNHX 5 – 6 Endosome

AtNHX 7 (SOS1) – 8 PM CPA = Cation / proton antiporter

© 2014 American Society of Plant Biologists

Loss of function of SOS1 makes

plants “salt overly sensitive”

Wu, S.J., Ding, L. and Zhu, J.K. (1996). SOS1, a Genetic Locus Essential for Salt Tolerance and Potassium Acquisition. Plant Cell. 8: 617-627. Shi, H., Ishitani, M., Kim,

C. and Zhu, J.-K. (2000). The Arabidopsis thaliana salt tolerance gene SOS1 encodes a putative Na+/H+ antiporter. Proc. Natl. Acad. Sci. USA. 97: 6896-6901.

Na+ H+

SOS1, aka NHX7, is a plasma-

membrane localized Na+/H+ exchanger

It has an auto-inhibitory domain that

can be phosphorylated to activate

the protein under salinity stress

© 2014 American Society of Plant Biologists

NHXs roles include Na+, K+ and H+

transport and homeostasis

Bassil, E., Coku, A. and Blumwald, E. (2012). Cellular ion homeostasis: emerging roles of intracellular NHX Na+/H+ antiporters in plant growth and development. J. Exp. Bot. 63: 5727-5740, by permission of Oxford University Press.

Na+ sequestration in vacuole Outward Na+ across PM

Regulation of pH of

endocytic

compartments for

proper protein

sorting and

modification

© 2014 American Society of Plant Biologists

Identification of salt tolerance in

halophytes and crop relatives

Chick pea

(Cicer arietinum)

See Flowers, T.J., Galal, H.K. and Bromham, L. (2010). Evolution of halophytes: multiple origins of salt tolerance in land plants.

Functional Plant Biology. 37: 604-612. Photo credits: Sanjay Acharya; Z. Hell; Fritz Geller-Grimm; Javier Martin, H2O

Salicornia spp.

Arthrocnemum

macrostachyum

Most sensitive Most tolerant

Glycophytes Halophytes

Salinity tolerance is a complex

trait and plants have a wide range

of salinity tolerances Halophytes are often defined as

having the ability to complete their

lifecycle on >200 mM NaCl

Wheat – intermediate sensitivity

(Triticum aestivum)

© 2014 American Society of Plant Biologists

Salt tolerance has evolved

repeatedly and independently

Flowers, T.J., Galal, H.K. and Bromham, L. (2010). Evolution of halophytes: multiple origins of salt tolerance

in land plants. Funct. Plant Biol. 37: 604-612; Bennett, T.H., Flowers, T.J. and Bromham, L. (2013). Repeated

evolution of salt-tolerance in grasses. Biol. Lett. 9: 20130029, by permission of the Royal Society.

RED indicates order that

includes some halophytes

All

vascu

lar

pla

nts

200 salt tolerant species

within the grasses, from

~76 independent events

© 2014 American Society of Plant Biologists

Halophytes can be grown on saline

soils for food and fodder

Image credits: M. Fagg, Australian National Botanic Gardens; Arizona State University. See Glenn, E.P., Anday, T., Chaturvedi, R., Martinez-Garcia, R., Pearlstein, S., Soliz, D., Nelson, S.G. and

Felger, R.S. (2013). Three halophytes for saline-water agriculture: An oilseed, a forage and a grain crop. Env. Exp. Bot. 92: 110-121;Flowers, T.J. and Colmer, T.D. (2008). Salinity tolerance in

halophytes*. New Phytol. 179: 945-963. Shabala, S. (2013). Learning from halophytes: Physiological basis and strategies to improve abiotic stress tolerance in crops. Ann. Bot. 112: 1209-1221.

Atriplex nummularia

(old man saltbush)

The genus Atriplex includes many

edible halophytes and is being

grown for fodder in Australia

Palmer’s grass (or nipa grass; Distichlis

palmeri) grows in tidal marshes of the Gulf

of California and was a food grain eaten by

the indigenous people of the region

Mex

USA

© 2014 American Society of Plant Biologists

Quinoa is a facultative halophyte

and a popular food grain

Bonales-Alatorre, E., Shabala, S., Chen, Z.-H. and Pottosin, I. (2013). Reduced tonoplast fast-activating and slow-activating channel

activity is essential for conferring salinity tolerance in a facultative halophyte, Quinoa. Plant Physiol. 162: 940-952. Maurice Chédel

Quinoa (Chenopodium quinoa) evolved in the

Andes and can tolerate saline soils

Young leaves (left) extrude salt into salt

bladders, older leaves (right) store it in vacuoles Quinoa is also a

useful model for

studies of salinity

tolerance

Vacuolar sodium channel activity is decreased in old

leaves (right) grown under salinity; no salinity effect is

observed in young leaves

Control

Salt

Control

Salt

© 2014 American Society of Plant Biologists

Models for salt tolerance:

Eutrema spp. (salt /saltwater cress)

Reprinted from Amtmann, A. (2009). Learning from evolution: Thellungiella generates new knowledge on essential and

critical components of abiotic stress tolerance in plants. Mol. Plant. 2: 3-12 by permission of Oxford University Press.

Arabidopsis thaliana

(Previously known as

Thellungiella halophila

or the related

Thellungiella

salsuginea)

Strategies for salt tolerance

include expansion of several gene

families (HKT, AVP) and lower

accumulation of Na+ in the shoot

as compared to Arabidopsis..

Higher selectivity

for K+ than Na+

uptake in the root

© 2014 American Society of Plant Biologists

Breeding and engineering for salt

tolerance

Reprinted from Roy, S.J., Negrão, S. and Tester, M. (2014). Salt resistant crop plants. Curr. Opin. Biotech. 26: 115-124.

Salt tolerance can

be attributed to

three non-

exclusive

mechanisms

Salinity

tolerance can

be enhanced by

breeding or

engineering

© 2014 American Society of Plant Biologists

Wheat yield on saline soils improved

by an ancestral Na+ transporter gene

Huang, S., Spielmeyer, W., Lagudah, E.S. and Munns, R. (2008). Comparative mapping of HKT genes in wheat, barley, and rice, key determinants

of Na+ transport, and salt tolerance. J. Exp. Bot. 59: 927-937 by permission of Oxford University Press; Credit: Dr Richard James, CSIRO

A pair of genes derived from a

relative of wheat confers enhanced

salinity tolerance

Tetraploid

pasta wheat

Hexaploid

bread wheat

Durum wheat carrying salt-

tolerance genes

Because these species are

closely related, the genes

can be introduced into

cultivated wheat without

using GM methods

© 2014 American Society of Plant Biologists

Nax1 and Nax2 exclude Na+ from leaf

blades by removal from xylem

Reprinted by permission from Macmillan Publishers Ltd from Munns, R., et al and Gilliham, M. (2012). Wheat grain yield on saline soils is improved by an ancestral Na+ transporter gene. Nat.

Biotech. 30: 360-364. Schroeder, J.I., et al and and Sanders, D. (2013). Using membrane transporters to improve crops for sustainable food production. Nature. 497: 60-66. Huang, S., Spielmeyer,

W., Lagudah, E.S. and Munns, R. (2008). Comparative mapping of HKT genes in wheat, barley, and rice, key determinants of Na+ transport, and salt tolerance. J. Exp. Bot. 59: 927-937.

When expressed in Xenopus

oocytes, the transporters

conduct Na+ but not K+

In plants, Nax1 and Nax2

pump Na+ into the cells

surrounding the xylem so it

does not reach the leaf blade

Nax1

Nax2

© 2014 American Society of Plant Biologists

The candidate gene approach has

had some success

Transgenic plants carrying vacuolar NHXs,

vacuolar H+-PPases and plasma membrane NHXs

have demonstrated enhanced salinity tolerance

H+

H+

H+ ATP ADP + Pi

ATP ADP+ Pi

PP 2 x Pi

Na+

H+

H+ Na+

SOS1

V-H+-ATPase H+-PPase NHX

PM-H+-ATPase

NSCC Na+

Na+ HKT

Enhanced ROS

detoxification and

synthesis of

compatible solutes

is also correlated

with enhanced

salinity tolerance

See for example Roy, S.J., Negrão, S. and Tester, M. (2014). Salt resistant crop plants. Curr. Opin. Biotechnology. 26: 115-124; Gaxiola, R.A., Li, J., Undurraga, S., Dang, L.M., Allen,

G.J., Alper, S.L. and Fink, G.R. (2001). Drought- and salt-tolerant plants result from overexpression of the AVP1 H+-pump. Proc. Natl. Acad. Sci. USA 98: 11444-11449; Apse, M.P.,

Aharon, G.S., Snedden, W.A. and Blumwald, E. (1999). Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis. Science. 285: 1256-1258.

© 2014 American Society of Plant Biologists

The intersection of potassium

nutrition and sodium toxicity

Cuin, T.A., Miller, A.J., Laurie, S.A. and Leigh, R.A. (2003). Potassium activities in cell compartments of salt‐grown barley leaves. J. Exp. Bot. 54: 657-661 with permission from Oxford University Press.

K+ext Na+

ext

K+ uptake

Na+ uptake

Na+ and K+ interfere with

each other’s uptake

When barley plants are grown

on 200 mM NaCl, they

accumulate Na+ at the

expense of K+ in their leaves

© 2014 American Society of Plant Biologists

As [Na+]ext increases and enters the

cell, K+ is driven out

H+

ATP

ADP + Pi

Non-selective

cation channel Na+

K+

Na+

Membrane

depolarizes

1. Steep concentration

gradient for Na+

2. Na+ leaks in

through NSCCs

3. Increased negative

charge within;

depolarized membrane

4. K+ driven out

through Kv channel

Some salt-tolerant plants

maintain elevated K+ by higher

activity of PM H+-ATPase

Chen, Z., Pottosin, I.I., Cuin, T.A., Fuglsang, A.T., Tester, M., Jha, D., Zepeda-Jazo, I., Zhou, M., Palmgren, M.G., Newman, I.A. and Shabala, S. (2007). Root plasma membrane transporters

controlling K+/Na+ homeostasis in salt-stressed barley. Plant Physiol. 145: 1714-1725; Shabala, S. and Cuin, T.A. (2008). Potassium transport and plant salt tolerance. Physiol. Plant. 133: 651-669.

Na+

K+

Furthermore,

Na+ directly

competes with

K+ for low- and

high-affinity

transporters

© 2014 American Society of Plant Biologists

Interaction between K+ nutrition and

Na+ toxicity

Cytosol

Vac.

Prioritized

Non-

Prioritized

K+ / Na+ ratio

K+ / Na+ ratio

Plants must

coordinate the

actions of K+ and Na+

transporters to

maintain a high ratio

of K+ to Na+ in

prioritized tissues

Adapted from Amtmann, A., and Leigh, R. (2010). Ion homeostasis. In Abiotic Stress Adaptation in Plants: Physiological, Molecular and Genomic

Foundation, A. Pareek, S.K. Sopory, H.J. Bohnert and Govindjee (eds) (Dordrecht, The Netherlands: Springer), pp. 245 – 262.

© 2014 American Society of Plant Biologists

Salinity tolerance: Summary

• Saline soils are detrimental to plants and

are widespread

• Sodium toxicity is primarily due to

interfering with K+ nutrition

• Sodium tolerance depends on exclusion,

extrusion an sequestration

• Breeding and engineering for salinity

tolerance have had mixed success so far

© 2014 American Society of Plant Biologists

Summary and ongoing research

K+

K+

PO43-

PO43-

PO43-

NO3-

NO3-

• Nutrient uptake is extremely energetically demanding

• Proton motive force generated by proton pumps is

essential for nutrient uptake

• Dozens of membrane transporters are involved in

uptake, allocation and homeostasis of mineral nutrients

• Most plants require a high cytosolic ratio of K+ to Na+

• Plants require large amounts of potassium for optimal

growth

• Sodium toxicity is a real and growing problem

• The mechanisms of sodium tolerance are being

identified and exploited for plant breeding