Investigation of the Genetic Control of Fibre Length in Arabidopsis thaliana through Gene Expression Profiling of the Intrusive Growth Phase of Interfascicular Fibres Hardy Hall PhD 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 plants Arabidopsis Poplar Hemp phloem xyleminterfascicular Fibre cells are key structural cells in vascular plants Mutations affecting fibre properties affect stem architecture ifl1 Fibres provide strength and elasticity Long Tapered 2 o cellwall (thick, lignified) Dead at maturity Fibres provide strength and elasticity Long Tapered 2 o cellwall (thick, lignified) Dead at maturity pith fibres High-rise rebar ≈ Bromeliad leaf South Dakota State Univ.W. Barthloltt, ‘83. Queens Univ. Burk, ‘02. Burk, ‘02 Zhong, ‘97

Mature fibre property determinants Cell expansion Cell wall fortification Cessation of elongation Developmental gradients Intrusive growth      Programmed cell death    

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 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 Length (mm) Lehle Ecotypes Length (mm) Frequency ABRC accessions

Arabidopsis is a model for studying fibre length Arabidopsis is a model for cell expansion Arabidopsis is a genetic model for complex traits Generation time Size Ploidy Compatibility Genome size Genetic and physical maps Functional annotation Knowledge base Oppenheimer (web) Zhang, ‘03Shimuzu, ‘00 Gunning (web)

Thesis 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 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 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 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 bamboo fibres Gritsch, ‘05 Ageeva, ‘05 Suh, ‘05

Establishing developmental equivalence Sources Genotype Microclimate Stochastic development Variable morphologies Bolt timing Growth rate Branch number Internode spacing 1. Internode # from SAM 2. Distance from SAM Sampling strategies 3. Cellular ontogeny dividingmature Hertzberg, ‘04

RNA amplification Biochemical analysis Expression profiling qPCR microarray sampling point Expansion rate (mm hr -1 ) 5 mm increments 3 Time (days) observation period Sampling schematic Chemical Fixation Cryostat Pooling 3 mm h -1 LCM 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 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 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

Timeline 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 RILs RIL 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 trials LCM trials qPCR follow-up Main Tasks Key Standardizations Determine intrusive growth timing/localization Find ultrastructure correlatives Epidermal cell study Locate intrusive growth events Stem 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 stems N=5 N=4 N=7 FQA analysis of 3-5mg samples 31% fines 28% fines 32% fines

Fibre length does not correlate with plant height