Hardy Hall Supervisor: Brian Ellis Co-supervisor: Carl Douglas

1. Annual Committee MeetingPresentationDetermining Genetic Regulation of Fibre Elongation in Arabidopsis thaliana Through Global Gene Expression Profiling Hardy Hall Supervisor: Brian Ellis Co-supervisor: Carl Douglas Committee: Lacey Samuels, Shawn Mansfield, Geoff Wasteneys Sept 22, 2008

2. Outline • Approaches to finding genes important in fibre development - a fibre-centric strategy • Program objectives - testable hypotheses • Progress update - from technical advancement to experiment • Workplan - from morphometrics to global gene expression profiling

3. Pulped fibres Fibre Review • Fibres are supportive cells with unique structure • Non-vascular • Thick secondary cell walls • Very long (>600um) • Very narrow (10-20um) • Tapered cell ends • Dead at maturity • Fibres occur in Arabidopsis: • Within xylem • Between vascular bundles • Fibre development is under genetic control courtesy of George Soong Transverse section IFF Xylary **What genes regulate fibre development and thus determine the morphology of mature fibres?

4. Approaches to Finding Genes Relevant to Fibre Development • Mutant screens for 'fibreless' phenotypes • Reverse genetics of putative fibre-specific genes • Quantitative genetics • Natural variation + aggressive phenotyping + genetic map = large effect loci • Global gene expression profiling (GGEP) • mRNA abundance shifts between distinct developmental states of fibres

5. Approaches to GGEP of Development • General approach: • Pool and replicate tissues of specific developmental state • Compare tissues of different developmental states • Isolate mRNA abundances that vary most markedly between those developmental states • Important GGEP Requirements • Refine developmental gradient as much as possible • Isolate large amounts of [transcriptionally active] tissue from a given replicate • Routinely identify of developmentally equivalent pools and replicate groups

6. Current GGEP Approaches • FACs sorting of cell types in roots (Benfey group) - Arabidopsis • Requires transgenic lines for every cell type • Requires separation of intact cell types • Post-GGEP tissue assignment (Sandberg group) - poplar • Precludes pooling of tissues • Requires large numbers of arrays for PCA • Partition tissues on 'rules of thumb' ( Douglas group) - Arabidopsis • Requires assumptions that harvested tissues accurately represent developmental states Which approach is suitable for Arabidopsis fibre transcriptomics?

7. Building Fibre Transcriptional Ontogeny in Arabidopsis • Challenges • Fibres are unique cells with unique genetic programs • Fibres traits are polygenic • Transcriptional changes are rapid and complex • Fibre development is poorly characterized in Arabidopsis (and other species) • Fibres grow within a challenging experimental context (inflorescence stems) • Small tissues require microscopic handling • Sample volumes are very low • Stem development is rapid and transient • Conclusion: • Focus on a few obvious morphological changes in fibres that are easy to track in planta • Sampling regime must exist that is both convenient and refined along this developmental gradient

8. Fibre Length is a Suitable Trait for Study by GGEP • Inflorescence stem expansion is developmental program of relative expansion rate of stem segments • Growth kinetic profiles (GKP) of stems define relationship of one part of the stem to another • Developmentally equivalent tissues could be routinely identified across plants on GKP data • Fibre morphology can be used to guide selection of tissue for fibre-specific GGEP study Movie (http://www.plantometrics.com/?q=node/201) Hypothesized GKP

9. Program Objectives • Hypothesis 1 (Morphometrics) • Fibres elongate in fixed relation to growth kinetic profiles of the Arabidopsis inflorescence stem • Hypothesis 2 (Transcriptomics) • Fibre-specific gene regulations can be detected from cell type-specific GGEP on growth kinetic profiles • Hypothesis 3 (Functional genomics) • GGEP data, public expression datasets, and collaborators' QTL datasets implicate common subset of large effect loci for fibre-specific elongation

10. Hypothesis 1 - GKPs and Fibre ElongationSpecific Examinations • Can GKPs for inflorescence stems be routinely identified? • Do parenchymal cell lengths correlate with GKPs? • Do fibres elongate in fixed relation with parenchymal cells? • If not, is there evidence for tip growth in cell wall modifications? • How consistent are these relations among replicate plants? • Can 'rules of thumb' for harvesting be validated and employed?

11. Hypothesis 1 -Growth Kinetic Profiling (GKP)Can GKPs be routinely determined? • Approach • Divide stems into a series of segments by synthetic optical markers (tags) • Monitor movement of tags over time (image capture) • Track tags across time series (image analysis) • Quantify stem segment relative growth rates by relative tag movement (growth kinetic analysis) 4 hour interval Movie (http://www.plantometrics.com/?q=node/205)

12. Hypothesis 1 - Growth Kinetic Profiling (cont.) GKPs can be routinely determined • Test case • Observe 6 Col-0 and 6 Ler plants at 10' intervals • Harvest at mid-day and determine GKPs • Observations • GKPs are consistent over time in a each plant • GKPs have sufficient detail to contrast >3 developmental states http://www.plantometrics.com/?q=node/240 http://www.plantometrics.com/?q=node/243 Landsberg erecta Columbia - 0

13. Hypothesis 1 - Growth Kinetic AnalysisImaging Improvements • Complete plant isolation • Linearized growth • Improved lighting

14. Hypothesis 1 - Growth Kinetic ProfilingAutomated Feature Tracking • Need for automation • High-throughput GKP for pooling • Near real-time GKPs for directed cell biology • Approach • Isolate tags from image by segmentation • Identify tag pairings • Calculate tag positions • Track tag movements over time

15. Hypothesis 1 - Morphometrics • Questions • Do parenchymal cell lengths correlate with GKPs? • Do fibres elongate in fixed relation with parenchymal cells? • If not, is there evidence for tip growth in cell wall modifications? • Approach • For ≥ 3 Col-0 plants: • Image and calculate GKPs • Harvest and preserve all segments • Perform morphometric analysis of fibres and other cell types (cell length and other?)

16. Published in planta longitudina fibre views Lev-Yadun, 1997 Zhong, 2006 Little, 2001 Spurs resin, 1um TEM Polarized light Wax-embedded, Safranin-fast green 1 mm Hypothesis 1 - Morphometric AnalysisHow can we measure fibres within stems? Problem: Fibre morphology defies conventional morphometrics • Mature cells are narrow (>10um wide) but very long (up to 1000um) • Sectioning is required to expose internal cells, including fibres • in planta fibre views are limited by: • Section orientation • Section thickness • Microscopic transparency of section • Histological preservation Sample Fibre from macerated tissue George Soong, 2005 brightfield Model based upon FQA data of Col-0 (90um) There is no existing method for routine in planta viewing of entire fibres (obtaining and viewing sections)

17. Hypothesis 1 - Morphometric AnalysisStep 1: Accurate determination of plane-of-section • Approach • Pins inserted into either end of 3mm segment in morphologically-equivalent radial position (within 75um radial tolerance) • Pin insertion points imaged for future reference • Top half stored for tissue imaging and morphometrics top bottom Sectioning tolerance for mature fibres

18. Progress on Hypothesis 1 - Morphometric AnalysisTangential sectioning now routine Demo movie • Staining • Imaging • Morphometrics

19. Progress on Hypothesis 1 - Morphometric AnalysisStep 2:Viewing sectioned tissues for morphometrics • Approach • Staining tissues with Congo Red (fluorescent) staining of tissues • Scanning tissue with LSCM (400X) over several millimeters (tiling) • Stitching of tiles for extended tissue reconstructions • Measuring fibres and other cell types from stitches Movie (http://www.plantometrics.com/?q=node/335)

20. Progress on Hypothesis 1 - Morphometric AnalysisSegment-wise cell measurements Measure how? • Use multiple slices of each confocal stitch to measure cell extremities either side of a periclinal transect Measure what? • Cortex - parenchymacollenchymal • "Endodermis" - parenchyma/procambium(?) • Fibre - parenchymasclerenchyma • Pith -parenchymasclerenchyma(?)

21. 1 cm Hypothesis 1 - MorphometricsFirst look at fibre elongation in context of GKP and diffuse elongation Cell counts Cortex = 20 Endodermis = 10 Fibre = ~4 Pith = ~20 Harvest image (2.5 hrs) overlayed upon Start image (11am)

22. Fold difference to cortex length Hypothesis 1 - MorphometricsPlant 1 Preliminary Analysis Expansion zone = 6 cm

23. Fold difference to cortex length Hypothesis 1 - MorphometricsPlant 2 Preliminary Analysis

24. Fall Plans • Complete morphometric examination • Improve sectioning and mounting procedures for morphometrics • Perform confocal tiling on 3-4 more plants (12 segments/plant) • Relate (statistically?) morphometrics and GKPs for 3-6 Col-0 plants • Look for evidence of [tip?] intrusive growth in fibres at specific GKP stages • Examination of cell expansion phenomena (XET/XTH activity, cytoplasmic streaming, etc…) in interfascicular fibre regions • Commence global gene expression profiling • Finalize microarray experiment (sample sizes, pooling regime, hyb pairings, statistical approach) • Amass Col-0 tissues for GGEP and cell biology • Optimize laser microdissection (LMD) and mRNA amplification