Kein Folientitel

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indole-3-acetic acid IAA
a naphthalene acetic acid NAA
2,4-dichlorophenoxyacetic acid 2,4-D
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control of plant development by auxin
embryo development
flowering
shoot branching
internode elongation
root branching
leaf growth
root elongation
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differentiation
elongation
division
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auxin
signal transduction
gene expression
cellular response
binding
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isolation of auxin signal transduction mutants
mutated gene
protein with lost or altered function
auxin
auxin perception and signal transduction
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selection of auxin-resistant mutants
(b.) mutagenized population of seedlings
(a.) wild-type seedlings
( - )
auxin
auxin
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auxin
signal transduction
gene expression
cellular response
binding
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signal transduction
gene expression
cellular response
binding
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Auxin-resistant mutants of Nicotiana
plumbaginifolia isolated by Petra Stirnberg and
Ann Blonstein
R15 looks like WT except for hyponastic
leaves, altered root gravitropism R25 extre
me dwarf, very wrinkled leaves, few lateral
roots, few root hairs, spontaneous callus
formation on old roots, late-flowering, dark
green, altered root gravitropism R28 short
plant, late-flowering, light green R29 rootless,
seedlings die young
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R15
WT
NAA
0 µM
3 µM
30 µM
100 µM
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Molecular Tools for Study of Auxin Action
SAUR-genes (Small Auxin Up-regulated RNAs) -
auxin-inducible within 2.5 min - primary
auxin-responsive genes - function unknown
GH3-genes - auxin-inducible in less than 20
min - primary auxin-responsive genes - function
unknown
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SAUR
GH3
Sven Borowski
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R15 mutant increased expression of SAUR mRNAs
Sven Borowski
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Projects available
Gene expression in auxin-resistant mutants

• Which of the auxin-related mutations affect
  rapid activation of auxin-inducible genes?
• Are only certain auxin-inducible genes
  affected?
• Techniques
• RT PCR
• Northern Blots
• RNAse protection assays

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auxin
signal transduction
gene expression
cellular response
binding
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analysis of auxin-induced promoter activation
isolate from plant
promoter
structural gene
fuse to reporter
promoter
reporter gene
re-introduce into plant analyze expression
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reporter genes for promoter activity
beta-glucuronidase (from E. coli)
Gus
visual inspection, microscopy
luciferase (from firefly)
Luc
luminometer, CCD-camera
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(No Transcript)
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Gus expression monitoring of tissue-and
cell-specific promoter activity
Gus expression controlled by GH3-promoter
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(No Transcript)
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visualizing auxin responses
auxin-treated
control
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SAUR-Gus Expression
WT
R15
0
0.01
1
100
NAA (µM)
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Luciferase expression monitoring promoter
activation kinetics
Laura Gutierrez
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Projects available
Activation of Auxin-inducible Promoters

• Influence of mutations in the auxin-response
  pathway (e.g. R15)
• pharmacological investigation of signal
  transduction
• Techniques
• Gus-staining ? cell-specificity of expression
• Luciferase ? kinetics of promoter activation

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Mycorrhiza and Auxin?

• mycorrhizal roots
• shorter, thicker, more branched
• increased auxin content
• auxin transport inhibitors change mycorrhizal
  development

? correlative evidence only
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Projects available
Isolation of Auxins from Mycorrhizal Fungi

• Screening for auxin-like compounds using
  auxin-inducible luciferase activity as a bioassay
• Isolation and characterization of compounds that
  activate the GH3 promoter
• Techniques
• Luciferase Assays ? identify auxin-like activity
• HPLC ? purify auxins prepare for identification

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Role of Peroxidase in Auxin-inducible Cell
Elongation

• One specific peroxidase isoenzyme is
  overexpressed in the auxin-resistant dgt mutant
  of tomato
• (Tricia Gearhart, 04)
• -gt likely important for auxin-responses or for
  cell elongation (or both)
• -gt What does this enzyme do in the wall?

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Projects available
Characterization of Tomato Cell Wall Peroxidase

• determine substrate-specificity of enzyme
• clone and characterize peroxidase genes
  expressed in tomato hypocotyls
• Techniques
• - Enzyme kinetics
• RT-PCR

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Projects available

• Gene expression in auxin-resistant mutants
• semi-quantitative RT-PCR
• Northern Blots
• RNAse protection assays
• Activation of auxin-inducible promoters
• beta-glucuronidase staining
• luciferase assays
• Isolation of auxins from mycorrhizal fungi
• auxin isolation, HPLC
• luciferase bio-assays
• Characterization of Tomato Cell Wall Peroxidase
• enzyme kinetics
• RT-PCR cloning