Functional genomic analysis of novel secondary cell wall genes in poplar (Populus trichocarpa)

Secondary cell walls (SCWs) contain a significant amount of fixed carbon that can be harnessed for the production of renewable energy. However, efficient conversion of wood-based biomass for use as an alternative fuel source is constrained by lignin, a phenolic polymer that is recalcitrant to enzymatic degradation. Many aspects of SCW biosynthesis remain enigmatic, including how genes of broad functional classes affect lignin content and composition. A genetic association mapping (AM) study in poplar (Populus trichocarpa) previously identified novel genes genetically associated with lignin trait variation. To test the hypothesis that these genes influence SCW biosynthesis, I screened 27 lignin-associated genes using in silico analyses and transfer-DNA (T-DNA) mutant phenotyping of Arabidopsis (Arabidopsis thaliana) homologs and identified two genes for in-depth functional characterization. First, Coiled-coil Protein of Unknown Function (CPU) was identified to be highly expressed in xylem and co-expressed with well-known SCW-related genes including SND1, a key transcriptional regulator of SCW biosynthesis in fibres. While AM predicted CPU to be significantly associated with total lignin content variation, this was not found in transgenic poplar over-expressing poplar CPU. Instead, transgenic poplars exhibited altered fibre length compared to wild-type. In Arabidopsis, a genetic interaction for CPU and Cellulose-Microtubule Uncoupling (CMU) was identified as the cpucmu1cmu2 triple mutant had decreased SCW thickening in fibre cells compared to wild-type, suggesting CPU to also influence microtubules during SCW deposition. Secondly, P. trichocarpa Nitrate Peptide Family 6.1 (PtNPF6.1), a member of the nitrate1/peptide transporter superfamily was characterized. PtNPF6.1 is expressed in the vascular tissue, as detected from transgenic PtNPF6.1pro:GUS lines. Transgenic poplar suppressed in PtNPF6.1 had elevated levels of total nitrogen corresponding to elevated levels of free glutamic acid and aspartic acid compared to wild-type. Under luxuriant nitrogen conditions, PtNPF6.1-suppressed lines produced wood with less syringyl lignin compared to wild-type. The findings suggest PtNPF6.1 may help regulate the long-distance transport of nitrogen. Altogether, previously unsuspected classes of genes identified through AM has broadened our understanding of genes that impact the cellular and physiological processes that contribute to wood formation which may enable further optimization of woody plants for a diversity of applications including bioethanol productionScience, Faculty ofBotany, Department ofGraduat