Plants make us happy
Dravigne, A., Waliczek, T.M., Lineberger, R.D., Zajicek, J.M. (2008) The effect of live plants and window views of green
spaces on employee perceptions of job satisfaction. HortScience 43: 183–187. Photo credit: tom donald
People at work
who can see
plants report
significantly
greater job
satisfaction than
those who can’t.
Drawing of cork by Robert Hooke, discoverer of “cells”
Cells were first observed in plants.
Photograph of cork cells
Photo credit: ©David B. Fankhauser, Ph.D
Mendel’s observation of peas
revealed the laws of inheritance
Experimental biology
Observation >
Description>
Question?>
Manipulation = Experiment >
Answering question (Understanding ?)
More questions
Experimental biology
Observation > Manipulation > Understanding
Money > Applications > Publishing
- Anatomy
- Physiology (spray and pray)
- Chemistry (identification of signals)
- Biochemistry (protein isolation/structure)
- Genetics (genes/mutants)
- Cell biology (subcellular structures)
- Molecular biology (gene manipulation)
Anatomy
PRIMING
EMERGENCE
Following process in time and with markers
Cell biology (subcellular structures)
Elution profiles of the purification of ACC oxidase activity from cherimoya fruit after passage through (A) Mono Q anion
exchange column, (B) Mono P chromatophocusing column, and (C) Sephadex G-75 gel filtration column (, ACC oxidase
activity; , protein).
Biochemistry
Chemistry (identification of signals)
extract Xwater
Physiology (spray and pray)
-nutrients (N,P,Ca,)
-conditions (water, light, stress)
-compounds (hormones, chemicals)
inhibitor stimulator
Genetics (genes/mutants)
Choice of research topic?
- Serendipity
-Model system
-Biological process
-Gene/Gene family
-Signaling pathway
-Available methods
-„Trendy topic“
Molecular biology
GENES
gene manipulation, tools to study gene functions
Arabidopsis thaliana
- Small, fully sequenced genome
- Easy genetics (diploid/self-polinater)
- Short vegetation time
- No large space requirement
- Simple organ and tissue structure
- Many established tools and facilities
(transformation, libraries, databases)
Genes
-choice (???)
-function - lost
- enhanced
- modulated
-expression - where (which tissue)
-control of expression (TF, upstream network)
- missexpression
-interacting network – look for partners
How to get your favorite gene?
- “Monte Carlo” candidate gene approach
- Functional complementation
- From the protein back to the gene
- Expression
- Forward genetics
“Lottery”
o Homology to known factors
(trimeric G-proteins)
o Interesting domains
(kinases, phosphatases)
o „Other“ reasons
(serendipity)
Functional complementation
Protein > gene
o Enzyme activity (CKX, ACS)
oLigand binding (affinity chromatography,
azidolabeling;
photoaffinity label azido-[3H]IAA - auxin binding proteins ABP1, Zm-
p60,
Cytokinin – cytokinin binding protein)
o Complex members
o Proteomics approaches
(differential display, phosphoproteomics,)
Differential Display,
Elution profiles of the purification of ACC oxidase activity from cherimoya fruit after passage through (A) Mono Q anion
exchange column, (B) Mono P chromatophocusing column, and (C) Sephadex G-75 gel filtration column (, ACC oxidase
activity; , protein).
Biochemistry
-Microsequencing
- Blast search:
amino acid > nucleotide
- Search for a gene
Protein > gene
Expression pattern
o Enhancer/Gene-trap libraries
o Differential display
substractive hybridisation
microarray
Gene and enhancer trap libraries
Microarray
Expression map of Arabidopsis root
Forward genetics
EMS mutagenesis
Mutant screen at seedling level
Patterning mutant types
Fatty acids
Sterols
Signalling
Vesicle traffic
Activation tagging - YUCCA
Second site mutagenesis - suppressors
Suppressors of CLV3 overexpression
Second site mutagenesis - enhancers
Chemical genetics
B. Screen for mutantsA. Screen for effective compound
Arabidopsis natural variations = natural mutants
QTL –quantitative trait locus
Gene verification
o Multiple alleles
o Transposone reversion
o Complementation
Towards a gene role
o Loss of function: Reverse genetics
o Gain of function: Ectopic expression
o Mosaics
o Sequence manipulations
o Phenotype analysis
o Biochemical function
Loss of function
o Reverse genetics/TILLING
o Antisense and RNAi approaches
o Immunomodulation
o Repression domain
o Titration
Reverse genetics
– indexed mutant
libraries
TILLING
Gain of function
o Overexpression
o Tissue specific expression
o Conditional expression
o Protein stabilisation
CaMV 35S Promotor
GENE of interest
Two component system for gene expression
The hidden function
of WUSCHEL
Mosaics – Cre/Lox
gene of interest
Mosaics – Cre/Lox
Ethanol inducible expression
The dexamethasone-inducible promoter
activating system
The tetracycline-inducible promoter
inactivation system
Sequence manipulation
o Site-directed mutagenesis
(phosporylation sites, activity of
protein domains, catalytic center)
o Domain deletions and swaps
o Chimeric proteins
rop GTPases mutants
rop GTPases mutants
AUX/IAA and ARF proteins
QVVGWPPVRSYRK
BDL
MP
S
Homo and hetero-
dimerisation
Protein
stability
Homo and hetero-
dimerisation
AuxRE binding
DBD MR III IV
IVIIIIII
ARF
Aux/IAA
bdl mutation
Hardtke and Berleth 1998
C2(366)C1
C2(505)C1
C1
C1C2
C2
Cry2502
Blue light photoreceptor-chimers
Cry1KO
Phenotype analysis
o Visual evaluation
o Ultrastructure (EMS)
o Use of markers
o Treatments
Biochemical function – test prediction
o Protein activity
o Yeast complementation
o Xenopus oocytes
0
0,5
1
1,5
2
2,5
3
relativeGDP/GTP
exchangerate
ARF
+GNOM
ARF
+GNOM
+BFA
ARFalone
Gene Expression and Protein
Localization
o Blots, RT-PCR
o Reporter genes
o In situ mRNA hybridization
o In situ protein localization
o In situ protein activity detection
Blots and RT-PCR
Northern blots RT-PCR
- IAA + IAA
Western blots
Reporter genes
o Transcriptional fusions
o Translational fusions
o GUS, Luciferase, GFP
o Applications
Transcriptional fusion
Translational fusion
GUS – ß-Glucuronidase
- Ferrocyanid + Ferrocyanid
GUS – ß-Glucuronidase
luciferin + O2 → oxyluciferin + light
Green Fluorescence Protein
Ca2+
Cell identity markers
Actin Tubulin
Subcellular structure markers
In situ mRNA/protein localisation
o Probe preparation
o Fixation
o Embedding
o Sectioning
o Deparafinization
o Treatment with probe
o Removal of unbound probe
o Signal visualization
GUS ProteinmRNA
Analysis of gene expression
Verifications? In-situ, immunolocalisations,
transcriptional fusion, translational fusions
Analysis of protein localisation
Friends and associates
o Yeast-two-hybrid
o Split ubiquitin, split YFP
o Genetic interactions
o Upstream and downstream
Classical transcription factor
1. DNA Binding domain
2. Activation domain
DNABinding
Activation
+
Bait Prey
Prey
lexA Protein VP16 Protein
Activation Activation
DNA-
binding
Yeast two hybrid
pSH18-34
genomic
lacZlexA Operator
lexA Operator
pSH18-34
genomic
lacZlexA Operator
lexA Operator Leu2
Leu2
DNA-
binding
Bait Activation
Bait
Summary for Y2H Prey
Prey
ActivationDNA-
binding
pSH18-34
genomisch
lacZlexA Operator
lexA Operator
pSH18-34
genomisch
lacZlexA Operator
lexA Operator Leu2
Leu2
Bait
Prey
(HIS3)
(TRP1)
(URA3)
lexA-BaitADH Promotor
VP16-PrayGal1 Promotor
EGY48: Mutant for HIS3, TRP1, URA3 und LEU2
Galaktose
Activation
Activation
DNA-
binding
DNA-
binding
Bait Prey
Conditions for Y2H-System
1. Proteins must be able to localize to the
nucleus
2. Bait construct must not have its own
activation domain
(Autoactivation)
Split-Ubiquitin
ubiquitin-specific
proteases (UBPs)
Co-immunoprecipitation
Split-YFP
o Protoplast transfection
c-Myc
P72S
Promoter (bp)
2,400 Yes
1,500 Yes
1,000 Yes
500 No
150 No
Activity
promoter::GUS
Upstream - Promotor analysis
(yeast one hybrid)
Yeast One-hybrid
• Identification of protein-promoter interactions
cDNA library
Prey
Activation domain
Transcription ON
Promotor
of interest Reporter gene
(HIS3 and LacZ)
Transcription OFF
Reporter gene
(HIS3 and LacZ)
Chromatin immunoprecipitation (ChIP)
Electrophoretic mobility shift assay (EMSA)
Co-expression
Transcription Factors – Targets
verification
Downstream targets
o expression profiling
o proteomics
o second site mutagenesis
o educated guess
Special methods and tools
o DR5 auxin response reporter
o Transient transfection
o Laser ablations and laser capture
DR5 (Auxin) Response Reporter
DR5 35S min GUS 35S pA
5´ CCTTT TGTCTC 3´
9x inv.
DR5: Ulmasov et al., 1997
- Auxin + Auxin
DR5::GUS
DR5::GFP Auxin Reporter
EmbryosRoot Root + Auxin
DR5rev 35S min GFP 35S pA
5´ CCTTT TGTCTC 3´
9x inv.
anti-IAA AB
Laser ablations
Transient transfection
Protoplasts
GUS GUS + Diphteria Toxin
Onion epidermis cells
GUS GUS + Diphteria Toxin
GUS + IPT (cytokinin biosynthesis)
Laser capture