Investigation of the Genetic Control of Fibre Length in Arabidopsis thaliana through Gene Expression...
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Investigation of the Genetic Control of Fibre Length in Arabidopsis thaliana through
Gene Expression Profiling of the Intrusive Growth Phase of
Interfascicular Fibres
Hardy HallPhD Prospect
July 6, 2006
Ph.D. Candidacy Examination
Outline
• Background– Why study fibres?– What regulates fibre growth?– How can we study complex traits?– Why study genetic regulation of fibre length
in Arabidopsis?
• Thesis objectives
• Work plan
• Timeline
• Conclusion
Fibres occur in many seed plantsFibres occur in many seed plants
Arabidopsis PoplarHemp
phloem xylem interfascicular
Fibre cells are key structural cells in vascular plants
Fibre cells are key structural cells in vascular plants
Mutations affecting fibre properties affect stem architecture
Mutations affecting fibre properties affect stem architecture
ifl1Fibres provide strength and elasticity• Long• Tapered• 2o cellwall (thick, lignified)• Dead at maturity
Fibres provide strength and elasticity• Long• Tapered• 2o cellwall (thick, lignified)• Dead at maturity
pith fibres
High-rise
rebar
≈
Bromeliadleaf
South Dakota State Univ. W. Barthloltt, ‘83. Queens Univ.
Burk, ‘02. Burk, ‘02 Zhong, ‘97
Mature fibre property determinantsMature fibre property determinants
• Cell expansion
• Cell wall fortification
• Cessation of elongation
• Developmental gradients
• Intrusive growth
• Programmed cell death
Systematic genetics approaches to fibre properties
Systematic genetics approaches to fibre properties
• Classical genetics (phenotype gene)• Mutant libraries + complex phenotyping
• Reverse genetics (gene phenotype)• NOT PRACTICAL
• Quantitative genetics (phenotype gene)• Natural variation + inbreeding (RILs) + genetic maps
• Expression profiling (gene - phenotype)• Natural variation + microarrays
• eQTL : Genetic maps + RILs + microarrays
• Classical genetics (phenotype gene)• Mutant libraries + complex phenotyping
• Reverse genetics (gene phenotype)• NOT PRACTICAL
• Quantitative genetics (phenotype gene)• Natural variation + inbreeding (RILs) + genetic maps
• Expression profiling (gene - phenotype)• Natural variation + microarrays
• eQTL : Genetic maps + RILs + microarrays
Model systems for studying fibre lengthModel systems for studying fibre length• Poplar and eucalyptus are model woody species• Poplar exhibits natural variation in fibre length• Poplar is a challenging genetic model• Arabidopsis exhibits natural variation in fibre length
0.3 0.5 0.7 0.9
Length (mm)1.1
18 Lehle Ecotypes
Length (mm)
30
20
10
0
Fre
quen
cy
0.4 0.6 0.8 1.0 1.2
150 ABRC accessions
Arabidopsis is a model for studying fibre lengthArabidopsis is a model for studying fibre length
• Arabidopsis is a model for cell expansion
• Arabidopsis is a genetic model for complex traits
Generation timeSize
PloidyCompatibilityGenome size
Genetic and physical mapsFunctional annotationKnowledge base
Oppenheimer (web) Zhang, ‘03 Shimuzu, ‘00 Gunning (web)
Thesis objectivesThesis objectives
• Identify milestone and gradients of fibre morphogenesis
• Characterize growth mode of fibres (intrusive?)
• Identify genes that regulate fibre length
• Characterize function of fibre length genes
• Identify milestone and gradients of fibre morphogenesis
• Characterize growth mode of fibres (intrusive?)
• Identify genes that regulate fibre length
• Characterize function of fibre length genes
Problem statementProblem statement
What are the genetic determinants of fibre length?
Ifl1/rev fra1,2,3
What genes affect fibre development?
C. Correlate ‘A’ and ‘B’ with stem morphometrics• Diffuse stem elongation rates, internode number, silique
emergence
Work plan 1. Determination of fibre developmental
gradients and milestones
Work plan 1. Determination of fibre developmental
gradients and milestones
B. Programmed cell death• Mitochondrial PT, vacuolar collapse, DNA fragmentation
A. Ontogenesis• Prophase-specific cyclin activity, mitotic figures, DNA replication
Dan, ‘03
Gunurwardena, ‘04
Work plan 2. Examination of mode of fibre cell expansion
Work plan 2. Examination of mode of fibre cell expansion
C. Less-destructive indicators of diffuse growth• Epidermal cells as diffuse/intrusive expansion indicators?• Live-cell imaging (non-transgenic approaches)
C. Less-destructive indicators of diffuse growth• Epidermal cells as diffuse/intrusive expansion indicators?• Live-cell imaging (non-transgenic approaches)
B. Investigate cell wall and cytoskeletal ultrastructure (evidence for diffuse or tip growth)
• Vesicle distribution, microtubule/microfilament dynamics, microfibril orientations
B. Investigate cell wall and cytoskeletal ultrastructure (evidence for diffuse or tip growth)
• Vesicle distribution, microtubule/microfilament dynamics, microfibril orientations
A. Identify intrusive growth events• Symplastic disruption and isolation, degradation of
middle lamella
A. Identify intrusive growth events• Symplastic disruption and isolation, degradation of
middle lamella
Symplastic continuity by plasmodesmata
Symplastic continuity by plasmodesmata
bamboofibres
Gritsch, ‘05
Ageeva, ‘05
Suh, ‘05
Establishing developmental equivalenceEstablishing developmental equivalenceSources
GenotypeMicroclimateStochastic development
Variable morphologiesBolt timingGrowth rateBranch numberInternode spacing
1. Internode #from SAM
2. Distancefrom SAM
Sampling strategies3. Cellular ontogeny
dividing mature
Hertzberg, ‘04
RNA amplification
Biochemical analysis
Expression profiling
qPCR microarray
samplingpoint
Expansion rate
(mm hr -1)
Quic
kTim
e™
an
d a
TIF
F (U
nco
mp
ress
ed
) d
eco
mp
ress
or
are
nee
ded
to s
ee t
his
pic
ture
.
5 mm increments
3
Time (days)
1 2 4 5 6 7 8
observation period
Sampling schematic
ChemicalFixation
Cryostat
Pooling
3 mm h-1
LCM
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
Epidermis (E)
Cortex (C)
Pith (P)
Interfascicular fibres (IFF)
Vascular bundle (VB)
LCM sampling
Segment sectioning
Segment selection
Epidermal cell length
Fibre length
Live cell imaging
Epidermal cell length
B. Global expression profiling (multi-factor)– Cell type– Developmental stage (expanding vs. fortifying)– Genotype (short- vs. long-fibred)
Work plan 3. Gene expression profiling
Work plan 3. Gene expression profiling
A. qPCR of known developmental markers– Correlate with fibre development milestones – Direct expression profile sampling
C. Follow-up qPCR– Validate interesting array expressions– Investigate candidate genes over wider factor range
D. Expression QTL– Correlate 30K oligo expression profiles with mapped markers
Ehlting, ‘05
Work plan 4. Functional characterization of candidate genes
Work plan 4. Functional characterization of candidate genes
B. Identify candidate genes• Bioinformatics (Genevestigator, AtGenExpress)
• Consensus QTL and expression profiling (eQTL)
B. Identify candidate genes• Bioinformatics (Genevestigator, AtGenExpress)
• Consensus QTL and expression profiling (eQTL)
C. Localization• Gene expression (promoter::GUS/xFP)• Protein expression (ORF::xFP)
C. Localization• Gene expression (promoter::GUS/xFP)• Protein expression (ORF::xFP)
A. Functional genomics• Clustering, PCA, gene ontologies, pathway-mapping
A. Functional genomics• Clustering, PCA, gene ontologies, pathway-mapping
Timeline20062006 20072007 20082008 20092009
Fibre cell wall status (microfibril angle, lignification)
Determine fibre developmental gradients/milestones Monitor stem expansion
Fibre origin - DAPI
Fibre death - TUNEL
Functional characterization of candidate genes
Identification of poplar homologs
Reverse genetics of candidate genes - intrusive events
Candidate gene selection
Conventional QTL of fibre length RILsRIL population generation
Fine-mapping
RILs Available
UPSC collaboration?
Tissue-specific expression profiling
eQTL (100 RILs, 1 stage)
Conventional microarrays (20 genotypes/2 stages)
qPCR marker survey
RNA amplification trialsRNA amplification trialsLCM trialsLCM trials
qPCR follow-up
Main Tasks
Key StandardizationsKey Standardizations
Determine intrusive growth timing/localization
Find ultrastructure correlativesEpidermal cell study
Locate intrusive growth events
Stem prep for confocal workStem prep for confocal work
Conclusions
1. Fibre length varies amongst natural accessions of Arabidopsis
2. Understanding the genetic regulation of fibre development requires systematic approach (QTL + expression profiling)
3. Description of fibre morphogenesis in Arabidopsis is novel and will help expression profiling
4. This project offers many opportunities to take advantage of new developments
Acknowledgements• Committee
– Brian Ellis (Supervisor, UBC)
– Carl Douglas (Co-supervisor, UBC)
– Lacey Samuels (Botany,UBC)
– Geoff Wasteneys (Botany,UBC)
– Shawn Mansfield (Forestry,UBC)
• Botany technical consultants– Eiko Kawamura (Botany, UBC)
– Minako Kaneda (Botany,UBC)
– David Johnston (Botany,UBC)
– Michael Friedman (Forestry, UBC)
• Substitute advisor – Ljerka Kunst (Botany,UBC)
• Collaborators– Rodger Beatson (BCIT/Forestry,UBC)
– Thomas Berleth (Botany, UofT)
– Richard Chandra (Forestry,UBC)
– Marcus Shi (Botany, UofT)
– Harry Chang (Forestry,UBC)
– George Soong (Forestry,UBC)
– Brian Poole (BCIT)
– Paul Bicho (Paprican)
• Personal support team- Noriko Tanaka (Home)
Khumbu ice falls, Mount Everest
Supplements
• Fibre length does not correlate with plant height
• Fibre length variation along mature Col-0 stems
• List of contingencies
Contingencies List
Problems Solutions
Fibre growth is diffuse Consider more general process (other cell types)
LCM is not possible Hand cut sectioning
RNA can’t be amplified reliably Pool tissue from replicates
Array lists are unintelligible Be pre-emptive, Do more bioinformatics!
Conventional QTL yields no candidates
Utilize published QTL data, UPSC?
Arabidopsis fibre length genes already found
Fibre growth already characterized Good! On to the arrays!
Fine! Lots of great fibre biology left to explore!
Fibre length variation along mature Col-0 stemsFibre length variation along mature Col-0 stems
N=5
N=5
N=4
N=7
FQA analysis of 3-5mg samples
31% fines
28% fines
28% fines
32% fines
Fibre length does not correlate with plant height
Fibre length does not correlate with plant height