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

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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