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Integrating Transport with Growth, Development & Adaptation

CBMG688R (Adv Plant Physiol Dev) Spring 2007

Heven Sze

Department of Cell Biology & Molecular Genetics

•Growth and development depends on the uptake, and translocation of mineral nutrients and metabolites.

• Signaling. Ion dynamics provide signals and act as 2nd messengers. Electrical, Ca, pH

•Movement – Turgor changes depend on osmotic changes & water transport. E.g. leaf movement, guard cell

•Development- hormone (e.g. auxin) transportPolarity

•Stress tolerance. Plants deficient in essential nutrients are susceptible to drought, cold, and pathogen.

•Survival. Plants can survive with v. low levels of nutrients and with v. high or toxic level of metals. I.e. plants can sense and respond. How? E.g. exclusion of toxic ions.

Integrating Transport with Plant Growth, Development and Survival

Picture taken from: Boyes et al. 2001. Plant Cell

Morning glory bloom depends on Na +/H+ exchanger.

Seedling growth depends on K+ uptake by AKT1 channel (Hirsch et al 98 Science)

Vac pH 6.6 vs 7.7Fukuda-Tanaka 2000 Nature

AKT1 takes up ion at low K+, 10-100 uM

Trans cyclooctene

Cu transporter is required for ethylene signalingRan1-1 mutant (Cu-ATPase) shows triple response in absence of ethylene. Hirayama et al. Ecker, 1999 Cell

Col, WT

ran1-1, 1-2 mutants

Eto1-5, ethylene overproducing mutant

Ctr1, constitutive triple response.

Interpret:

Major Basic Questions

• Three classes of membrane transporters

• A. Three types: pumps, cotransporters, channels

• What do they do in cells?

• How are they studied?

• How do developmental cues and environmental signals influence the expression and activity of transporters?

e.g. How does hormone transport affect growth and development?: auxin (next time)

outline• Macro- and micronutrients• Distinction between pumps, cotransporters, and

channels• H+ extrusion pumps generate a H+ electrochemical

gradient in plant cells• H+ electrochemical gradient provides energy

to drive co-transport• Ions flow downhill via channels• Nernst equation predicts ion conc. at equilibrium• Methods to study each• Period of ‘omics’- functional genomics,

transcriptomics• Ions & signaling: e.g. Guard cells, pollen tube

growth

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Table 5.1 Taiz. Essential nutrients required by plantsA. Macronutrient Available Conc in Dry matter

Form umol/g µM [medium]

H H2O 60,000

C CO2 40,000

O O2, CO2 30,000

N NO3-, NH4

+ 1,000 16,000

K K+ 250 6,000

Ca Ca + + 125 4,000

Mg Mg + + 80 1,000

P H2PO4-, HPO4

-- 60 2,000

S SO4-- 30 1,000

Micronutrients

B. Micronutrient Available Form umol/g µM

Cl Cl- 3.0 50

B BO3- 2 25

Mn Mn2+ 1 2

Zn Zn2+ 0.3 2

Cu Cu2+ 0.1 0.5

Mo MoO4 - 0.001 0.5

Fe Fe2+ 1.0-3.0 16-50 Optional

Ni Ni2+ 0.02 0.5

Si Si 28 1,000

Na Na+

+ 1 mM Pi

-Pi starved

0 mM

Pi starvation induces lateral root growth-Lupinus

Plants sense nutrient quality and quantity, and then respond Cells regulate their intracellular ion

contentPlant Cell

Cytosol: pH 7.3

75-100 mM K

8 mM Na

10 mM Cl

3 mM Mg

< 1 µM Ca

-140 mV

Lodish. Fig. 5-43

Outside

pH 5.5

1 mM K

1 mM Na

1 mM Cl

0.25 mM Mg

1 mM Ca

3 H+ pumps generate electrical potentials and pH gradientsin plant cells:

-140 mV inside (cyt/ext)-110 mV (vac/outside)

H+

H+

A

B

H+V-ATPase

PPaseH+

H+

A

B

H+

pH 7.3

-140 mV

pH 5.5

+30 mV

pH 5.5

PM H+-ATPase

1970s, electrophysiol1982-1986, vesicles

Vac

H+ electrochemical gradient provides energy to drive co-transport

and ion flow via channels

H+

H+

H+

S

BAntiport

Symport

How will uptake of S affect the membrane potential?

pH gradient?

H+

C+

A-Ion Channels

3

Electrical potential affects the direction of ion flow

pH gradient is used to drive H+-coupled cotransporters

•Passive transport is movement down an electrochemical gradient.•Channels•Porters

•Active transport is movement against an electrochemical gradient.•Pumps•cotransporters

----------------------------------------------------------------------------------------Ext Conc. Ion Internal concentration (mM)

observed Nernst (Predicted)----------------------------------------------------------------------------------------

1 mM K+ 75 mM 74 (near equilibrium)1 mM Na+ 8 mM 74 (not at equilbrium)

1 mM Ca2+ 2 mM 5,0000.2 mM Mg2+ 3 1,340

2 mM NO3 - 5 mM 0.02

1 Cl- 10 mM 0.011 H2PO4

- 21 0.01----------------------------------------------------------------------------------------

At 1 mM Kext, K+ distributes according to Nernst prediction

Tab 6-1, Taiz . Nernst equation predicts ion conc. at equilibrium.e.g. Cell electrical potential, ∆ψ = -110 mV

Summary: In general

Cation uptake: passive

Cation efflux: active

Anion uptake: active

Anion release: passive

Fig. 6-4, Taiz. Passive and active fluxes can be predicted from the Nernst equation. Dawn of genomics in plants

Dec. 2000

Arabidopsis thaliana genome is completely sequenced!

Cover of Nature & Science. On Dec 14th, 2000, the Arabidopsis genome sequencing project is published in Nature

We celebrated with a party!

A revolution.

Changes the way we think and do research.

New skills and approaches are emerging.

Transporters make up ~5% of the total genes in

Arabidopsis (2000).

~1000 Transporters include:Pumps- primary transportCotransporters: secondaryChannels- passive

Bulk of transporters (60%) are secondary. e.g. Cation/H+ exchangers or

symporters

H+

Na/K+

pH 5.5

Cl-

NO3

Vac

ATP

H+

From TAGI 2000. Nature 408, 796

Substrates

45 P-type ionATPases in Arabidopsis

-------

PUMPS

Axelsen & Palmgren 2001 Plant Physiol

Heavy Metal

PM H+

ATPaseCa2+-ATPase

Amino-phospholipid

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Phylogeneticanalysis ofArabidopsistransporters

is used to infer function.

Sanchez-Fernandez et al. 2001. JBC

ABC protein

sequences120

How do you determine the specific ion or metabolite transported?Methods to study a transporter and determine its cellular roles

1. Assay Transport activity- in whole cell or isolated membrane/organelle (in vitro)a. isotope flux, b. current (channel)c. Flourescence dye (pH, electrical, Ca)

2. Distinguish one type of transporter from another using a. Specific inhibitorsb. Energy source: is it a pump or cotransporter. Ionophores dissipate ion gradient

3. Identify protein & its activity and the functional domainsa. Biochemical- purify and reconstitute in simple system-b. Molecular- Clone gene- express cDNA/gene in heterologous systemMutate or delete functional domains or residues

4. Determine role of transporter in whole organism (in vivo)a. Localize it to membrane, cell-type, and tissue

Immuno-detection, epitope Tag , GFP-tag

b. Test mutant for what is impaired. Reverse genetics: What cellular activity is changed in a mutant? E.g. signal transduction pathways

5. Application . Test if its overexpression or reduced expression in a organism or cell gives desired property. E.g. tolerance to toxic compounds.

Functional expression if you have the gene. [1992-now]

Test function by expression in a heterologous system.

----------------------------------------1. Obtain cDNA encoding a putative transporter

2. Express in a heterologous system

e.g. a suitable yeast mutant, COS cell

3. Test for rescue of wild-type phenotype

4. If transporter is localized on PM, measure uptake of radioactive S into yeast.

5. If transporter is localized on endomembrane, isolate membrane vesicle to measure transport.

cDNA -transporter

•Bacteria,

•Yeast mutant,

•frog oocyte,

•COS Tissue-culture cell

Test plant Ca pumps in yeast mutant.Yeast mutant grows poorly on low Ca media.

Plant gene restores wild-type growth

Test AtECA1 function.AtECA1 restores yeast mutant growth on 1 µM Ca

Liang F. et al 1997. PNAS

Empty vector

vector-ECA1

Yeast Cells

Lysate

Pel Sup

20%

45%Sucrose

Membrane

Sup45Ca2+

45Ca2+

H+V-ATPase

Ca-ATPase

Vanadate

BafilomycinGramicidinor CCCP

CAX1Antiport

Isolation of Membrane Vesicles 45Ca transport

ATP

ATP

Vesicles are collected on filters,and 45Ca is counted.

Test activity directly in isolated membrane

5

N-terminal truncated ACA2-2 is an active Ca2+ pump

Calmodulin stimulated the full-length ACA2-1 pump but not the

N-terminal truncated ACA2-2

Determine functional or regulatory domains of protein

ACA2-1

ACA2-2 Hwang et al 2000. Plant Physiol

Experiments to show H+-coupled cotransporters

e.g. PM H+/glucose symporter

H+/sucrose symporter expressed in yeast

Vac H+/Na+ exchanger

6-11 Taiz. Evidence that Glucose uptake depends on H+-coupled glucose symport

Functional studies with oocyte

1. Patch-clamp to measure changes in current

2. Direct transport assay using isotope

Ion ChannelsFig. 6-8. K+ enters cells via a gated channel.

K+

Channels are gated by voltage, Ligands: Ca, hormone, ATP, Glutamate Hyperpolarization-act

Depolarization-act

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Box6-1 Taiz. Measuring ion fluxes via channels with patch clamp method. Measures current (movement of a + charge)

Voltage-gated current. Evidence for K+ conductance

18-8. Stomata. Open and closed state

What controls opening? How? Increase in turgor pressure

What controls closing?

From Schroeder and Allen. 2001. Nature

Closing stomatal aperture: the long-term efflux of both anions and K+ from guard cells contributes to the loss of guard cell turgor, leading to stomatal closing.

Pollen development, growth & guidance[Johnson M & E Lord 2006] Ca affects polarized

tip growth in pollen