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 2 nd messengers. Electrical, Ca, pH •Movement – Turgor changes depend on osmotic changes & water transport. E.g. leaf movement, guard cell •Development- hormone (e.g. auxin) transport Polarity •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 7.7 Fukuda-Tanaka 2000 Nature AKT1 takes up ion at low K+, 10-100 uM Trans cyclooctene Cu transporter is required for ethylene signaling Ran1-1 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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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
•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
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
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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