Finding New Cell Wall Regulatory Genes in Populus trichocarpa Using Multiple Lines of Evidence.

Understanding the regulatory network controlling cell wall biosynthesis is of great interest in Populus trichocarpa, both because of its status as a model woody perennial and its importance for lignocellulosic products. We searched for genes with putatively unknown roles in regulating cell wall biosynthesis using an extended network-based Lines of Evidence (LOE) pipeline to combine multiple omics data sets in P. trichocarpa, including gene coexpression, gene comethylation, population level pairwise SNP correlations, and two distinct SNP-metabolite Genome Wide Association Study (GWAS) layers. By incorporating validation, ranking, and filtering approaches we produced a list of nine high priority gene candidates for involvement in the regulation of cell wall biosynthesis. We subsequently performed a detailed investigation of candidate gene GROWTH-REGULATING FACTOR 9 (PtGRF9). To investigate the role of PtGRF9 in regulating cell wall biosynthesis, we assessed the genome-wide connections of PtGRF9 and a paralog across data layers with functional enrichment analyses, predictive transcription factor binding site analysis, and an independent comparison to eQTN data. Our findings indicate that PtGRF9 likely affects the cell wall by directly repressing genes involved in cell wall biosynthesis, such as PtCCoAOMT and PtMYB.41, and indirectly by regulating homeobox genes. Furthermore, evidence suggests that PtGRF9 paralogs may act as transcriptional co-regulators that direct the global energy usage of the plant. Using our extended pipeline, we show multiple lines of evidence implicating the involvement of these genes in cell wall regulatory functions and demonstrate the value of this method for prioritizing candidate genes for experimental validation

Large-Scale Co-Expression Approach to Dissect Secondary Cell Wall Formation Across Plant Species

Plant cell walls are complex composites largely consisting of carbohydrate-based polymers, and are generally divided into primary and secondary walls based on content and characteristics. Cellulose microfibrils constitute a major component of both primary and secondary cell walls and are synthesized at the plasma membrane by cellulose synthase (CESA) complexes. Several studies in Arabidopsis have demonstrated the power of co-expression analyses to identify new genes associated with secondary wall cellulose biosynthesis. However, across-species comparative co-expression analyses remain largely unexplored. Here, we compared co-expressed gene vicinity networks of primary and secondary wall CESAsin Arabidopsis, barley, rice, poplar, soybean, Medicago, and wheat, and identified gene families that are consistently co-regulated with cellulose biosynthesis. In addition to the expected polysaccharide acting enzymes, we also found many gene families associated with cytoskeleton, signaling, transcriptional regulation, oxidation, and protein degradation. Based on these analyses, we selected and biochemically analyzed T-DNA insertion lines corresponding to approximately twenty genes from gene families that re-occur in the co-expressed gene vicinity networks of secondary wall CESAs across the seven species. We developed a statistical pipeline using principal component analysis and optimal clustering based on silhouette width to analyze sugar profiles. One of the mutants, corresponding to a pinoresinol reductase gene, displayed disturbed xylem morphology and held lower levels of lignin molecules. We propose that this type of large-scale co-expression approach, coupled with statistical analysis of the cell wall contents, will be useful to facilitate rapid knowledge transfer across plant species

Transcriptional Regulation of Sorghum Stem Composition : Key Players Identified Through Co-expression Gene Network and Comparative Genomics Analyses

Most sorghum biomass accumulates in stem secondary cell walls (SCW). As sorghum stems are used as raw materials for various purposes such as feed, energy and fiber reinforced polymers, identifying the genes responsible for SCW establishment is highly important. Taking advantage of studies performed in model species, most of the structural genes contributing at the molecular level to the SCW biosynthesis in sorghum have been proposed while their regulatory factors have mostly not been determined. Validation of the role of several MYB and NAC transcription factors in SCW regulation in Arabidopsis and a few other species has been provided. In this study, we contributed to the recent efforts made in grasses to uncover the mechanisms underlying SCW establishment. We reported updated phylogenies of NAC and MYB in 9 different species and exploited findings from other species to highlight candidate regulators of SCW in sorghum. We acquired expression data during sorghum internode development and used co-expression analyses to determine groups of co-expressed genes that are likely to be involved in SCW establishment. We were able to identify two groups of co-expressed genes presenting multiple evidences of involvement in SCW building. Gene enrichment analysis of MYB and NAC genes provided evidence that while NAC SECONDARY WALL THICKENING PROMOTING FACTOR NST genes and SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN gene functions appear to be conserved in sorghum, NAC master regulators of SCW in sorghum may not be as tissue compartmentalized as in Arabidopsis. We showed that for every homolog of the key SCW MYB in Arabidopsis, a similar role is expected for sorghum. In addition, we unveiled sorghum MYB and NAC that have not been identified to date as being involved in cell wall regulation. Although specific validation of the MYB and NAC genes uncovered in this study is needed, we provide a network of sorghum genes involved in SCW both at the structural and regulatory levels

THE REGULATORY MECHANISM OF SECONDARY CELL WALL BIOSYNTHESIS IN GRASSES

Grass cell walls are environmentally and economically important, including being an abundant and sustainable carbon source to produce lignocellulosic biofuels. However, the crosslinked structure of cell walls limits polysaccharide extraction efficiency, which is a bottleneck for biofuel production. Based on knowledge in Arabidopsis, multiple transcription factors from various protein families can regulate cell wall biosynthesis by forming a series of feed-forward loops. Diverged from dicotyledonous plants approximately 150 million years ago, grasses have evolved different cell wall components and vascular bundle patterning in vegetative organs. In this dissertation, I aimed to characterize transcription factors and corresponding DNA binding sites that control cell wall biosynthesis in grasses. I hypothesized that unstudied grass cell wall transcription factors might fall into the following three categories: (1) orthologs of known dicot cell wall regulators that have conserved functions in regulating the cell wall network; (2) uncharacterized cell wall-associated transcription factors that also likely maintain similar functions with those in dicots; (3) uncharacterized grass cell wall-associated transcription factors that do not exist or have different functions in dicots. In Chapter 2, to analyze conservation and divergence between known dicot cell wall-associated transcription factors and their orthologs in grasses, we examined the phylogeny of R2R3 MYB protein family across selected dicots and grasses. Though we observed dicot-specific, grass-specific, and two panicoid grass-expanded clades, in general, most R2R3 MYBs that regulate SCW in Arabidopsis show evidence of conservation in the grasses. In Chapter 3, we developed a Rice Combined mutual Ranked (RCR) network to identify regulators of grass-specific genes and other uncharacterized cell wall-associated transcription factors in grasses. The RCR network covers approximately 90% of the rice genome and shows high quality in GO-term-based evaluations. Network prediction and further molecular genetic validation suggest that OsMYB61a can directly or indirectly regulate grass cell wall-specific genes, among others. The RCR network includes a cell wall sub-network with 96 novel transcription factors. Eight out of eleven of them altered expression of cell wall-related genes in a transient gene expression assays in rice protoplast. In Chapter 4, I further examined the conservation of cell wall-associated cis-elements in grasses using comparative de novo motif discovery and explored various scenarios for incorporation of grass-specific genes into cell wall biosynthesis pathways. Firstly, we observed that known dicots cell wall-associated cis-elements, such as MYB and NAC DNA binding sites, are significantly enriched within the promoters of CESA, lignin biosynthesis genes, as well as grass cell wall-specific genes. This provides support for the generally held hypothesis that known dicot cell wall-associated cis-elements are conserved in grasses. In addition, cis-elements that are potentially associated with AP2/ERF, C2H2, C2C2, and homeodomain proteins are also significantly enriched within promoters of grass cell wall biosynthesis genes. These results support the prediction and characterization of novel cell wall-associated transcription factors and binding sites. In all, this dissertation provides guidance toward functional characterization of cell wall-associated regulatory elements in grasses, knowledge of which will promote terrestrial biofuel production

Pleiotropic and Epistatic Network-Based Discovery: Integrated Networks for Target Gene Discovery

Biological organisms are complex systems that are composed of functional networks of interacting molecules and macro-molecules. Complex phenotypes are the result of orchestrated, hierarchical, heterogeneous collections of expressed genomic variants. However, the effects of these variants are the result of historic selective pressure and current environmental and epigenetic signals, and, as such, their co-occurrence can be seen as genome-wide correlations in a number of different manners. Biomass recalcitrance (i.e., the resistance of plants to degradation or deconstruction, which ultimately enables access to a plant’s sugars) is a complex polygenic phenotype of high importance to biofuels initiatives. This study makes use of data derived from the re-sequenced genomes from over 800 different Populus trichocarpa genotypes in combination with metabolomic and pyMBMS data across this population, as well as co-expression and co-methylation networks in order to better understand the molecular interactions involved in recalcitrance, and identify target genes involved in lignin biosynthesis/degradation. A Lines Of Evidence (LOE) scoring system is developed to integrate the information in the different layers and quantify the number of lines of evidence linking genes to target functions. This new scoring system was applied to quantify the lines of evidence linking genes to lignin-related genes and phenotypes across the network layers, and allowed for the generation of new hypotheses surrounding potential new candidate genes involved in lignin biosynthesis in P. trichocarpa, including various AGAMOUS-LIKE genes. The resulting Genome Wide Association Study networks, integrated with Single Nucleotide Polymorphism (SNP) correlation, co-methylation, and co-expression networks through the LOE scores are proving to be a powerful approach to determine the pleiotropic and epistatic relationships underlying cellular functions and, as such, the molecular basis for complex phenotypes, such as recalcitrance

Organellar carbon metabolism is co-ordinated with distinct developmental phases of secondary xylem

Subcellular compartmentation of plant biosynthetic pathways in the mitochondria and plastids requires coordinated regulation of nuclear encoded genes, and the role of these genes has been largely ignored by wood researchers. In this study, we constructed a targeted systems genetics coexpression network of xylogenesis in Eucalyptus using plastid and mitochondrial carbon metabolic genes and compared the resulting clusters to the aspen xylem developmental series. The constructed network clusters reveal the organization of transcriptional modules regulating subcellular metabolic functions in plastids and mitochondria. Overlapping genes between the plastid and mitochondrial networks implicate the common transcriptional regulation of carbon metabolism during xylem secondary growth. We show that the central processes of organellar carbon metabolism are distinctly coordinated across the developmental stages of wood formation and are specifically associated with primary growth and secondary cell wall deposition. We also demonstrate that, during xylogenesis, plastid-targeted carbon metabolism is partially regulated by the central clock for carbon allocation towards primary and secondary xylem growth, and we discuss these networks in the context of previously established associations with wood-related complex traits. This study provides a new resolution into the integration and transcriptional regulation of plastid- and mitochondrial-localized carbon metabolism during xylogenesis

Finding New Cell Wall Regulatory Genes in Populus trichocarpa Using Multiple Lines of Evidence

Understanding the regulatory network controlling cell wall biosynthesis is of great interest in Populus trichocarpa, both because of its status as a model woody perennial and its importance for lignocellulosic products. We searched for genes with putatively unknown roles in regulating cell wall biosynthesis using an extended network-based Lines of Evidence (LOE) pipeline to combine multiple omics data sets in P. trichocarpa, including gene coexpression, gene comethylation, population level pairwise SNP correlations, and two distinct SNP-metabolite Genome Wide Association Study (GWAS) layers. By incorporating validation, ranking, and filtering approaches we produced a list of nine high priority gene candidates for involvement in the regulation of cell wall biosynthesis. We subsequently performed a detailed investigation of candidate gene GROWTH-REGULATING FACTOR 9 (PtGRF9). To investigate the role of PtGRF9 in regulating cell wall biosynthesis, we assessed the genome-wide connections of PtGRF9 and a paralog across data layers with functional enrichment analyses, predictive transcription factor binding site analysis, and an independent comparison to eQTN data. Our findings indicate that PtGRF9 likely affects the cell wall by directly repressing genes involved in cell wall biosynthesis, such as PtCCoAOMT and PtMYB.41, and indirectly by regulating homeobox genes. Furthermore, evidence suggests that PtGRF9 paralogs may act as transcriptional co-regulators that direct the global energy usage of the plant. Using our extended pipeline, we show multiple lines of evidence implicating the involvement of these genes in cell wall regulatory functions and demonstrate the value of this method for prioritizing candidate genes for experimental validation

An Integrative Approach to the Identification of Arabidopsis and Rice Genes Involved in Xylan and Secondary Wall Development

Xylans constitute the major non-cellulosic component of plant biomass. Xylan biosynthesis is particularly pronounced in cells with secondary walls, implying that the synthesis network consists of a set of highly expressed genes in such cells. To improve the understanding of xylan biosynthesis, we performed a comparative analysis of co-expression networks between Arabidopsis and rice as reference species with different wall types. Many co-expressed genes were represented by orthologs in both species, which implies common biological features, while some gene families were only found in one of the species, and therefore likely to be related to differences in their cell walls. To predict the subcellular location of the identified proteins, we developed a new method, PFANTOM (plant protein family information-based predictor for endomembrane), which was shown to perform better for proteins in the endomembrane system than other available prediction methods. Based on the combined approach of co-expression and predicted cellular localization, we propose a model for Arabidopsis and rice xylan synthesis in the Golgi apparatus and signaling from plasma membrane to nucleus for secondary cell wall differentiation. As an experimental validation of the model, we show that an Arabidopsis mutant in the PGSIP1 gene encoding one of the Golgi localized candidate proteins has a highly decreased content of glucuronic acid in secondary cell walls and substantially reduced xylan glucuronosyltransferase activity