Investigations into the transcriptional regulation of wood formation
AffiliationSchool of Ecosystem and Forest Sciences
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2022-11-13. This item is currently available to University of Melbourne staff and students only, login required.
© 2020 Narmada Nadeeshani Hetti Karannagoda
Xylogenesis (wood formation) is one of the planet’s most crucial biological processes. It is characterised by the complex, multi-stage development of cells commencing with periclinal divisions of cambial initials which subsequently undergo a series of dynamic, differentiation stages including further cell division, xylem cell expansion, secondary cell wall deposition and programmed cell death. Each of these stages is orchestrated by molecular controllers which are strictly regulated by an underlying network of transcription factors, the central regulators of wood formation. These transcription factors determine the morphology, chemical composition and physical properties of xylogenic cells as well as the overall dynamics of the wood formation process including its responsiveness to biotic and abiotic stresses including distinct responses to gravitational stress. This thesis used wood formed in response to gravitational stress (reaction wood) to dissect the transcriptional regulation of xylogenesis in order to gain detailed insights into the molecular control of wood formation in woody perennials. It does this by examining the current literature on transcriptional regulation of wood and reaction wood formation with specific focus on the roles played by transcription factors. Using this information, nine candidate transcription factors were chosen for subsequent experimentation to add to our current sketchy understanding of their specific functions during wood formation. Selected genes from Eucalyptus included INDOLE-3-ACETIC ACID INDUCIBLE 13 (EgIAA13), KNOTTED-LIKE HOMEODOMAIN 7 (EgKNAT7), MYELOBLASTOSIS 103 (EgMYB103), SECONDARY WALL NAC DOMAIN 2 and 3 (EgSND2 and EgSND3), BELL1-LIKE HOMEODOMAIN 6 (EgBLH6), EgWRKY2 and EgWLIM1. Aiding these functional investigations, also a suite of contemporary microanalytical techniques was reviewed assessing their suitability for phenotyping wood cell morphology and cell wall chemical composition in instances where available wood samples amount to as little as a only a few micrograms, as was the case for the induced somatic sector analysis (ISSA) derived samples analysed in this thesis. In these in vivo transformation experiments, up- and down-regulation constructs of candidate transcription factors were used to perturb wood formation in the ecologically and economically important forest tree species Eucalyptus and Populus. Our investigations revealed that EgIAA13 significantly alters xylem fibre and vessel morphology as well as xylem cell division rates, identifying EgIAA13 as a novel regulator of cambium dynamics and xylem formation. Subsequent protein-protein interaction analyses revealed that EgIAA13-EgARF (2, 5, 6 and 19) modules are presumably involved in mediating this dual regulatory role of EgIAA13. Our results also demonstrate that EgKNAT7 and EgMYB103 significantly alter xylem fibre morphology in terms of secondary cell wall thickness and fibre size, confirming their regulatory involvement in mediating secondary cell wall deposition and xylem expansion, respectively.
KeywordsEucalyptus, xylogenesis, secondary growth, secondary xylem, wood, transcription factor, transcriptional regulation, auxin, Induced Somatic Sector Analysis, phenotyping, small sample, microscopy, spectroscopy, morphology, xylem fibre, xylem vessel, EgIAA13, EgKNAT7, EgMYB103, tension wood
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