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Enhanced Resistance Pines for Improved Renewable Biofuel and Chemical Production (Technical Report)
We completed phenotyping constitutive and inducible oleoresin flow across two seasons, constitutive resin canal number and density and wood terpene content in our ADEPT2 and CCLONES populations. We completed genetic association between 19 oleoresin phenotypes and a total of 523,192 SNP markers from ADEPT2 and 13,883 SNP markers in CCLONES using four mixed linear models. A total of 293 significant SNPs (FDR = 0.20) were identified. We used the MENTOR tool to mine mechanistic connections from a multiplex network constructed from poplar multi-omic data to construct a conceptual model for a subset of these significant SNPs. Our model contains 6 transcriptional regulators in addition to 3 monoterpene synthases. To generate more lines of evidence for these significant SNPs, we completed a time course RNAseq experiment after inducing vascular zone cells to differentiate into new resin canals with a methyl jasmonate treatment, a single nuclei RNAseq that identified differentiating resin canal epithelial cells and are completing analysis for a QTL study in a hybrid pine population. The time course identified 4634 significantly down and 1890 significantly up regulated transcripts after treatment with methyl jasmonate, an inducer of new resin canal formation in the vascular cambial meristem. To analyze this large set of differentially regulated genes, we created a predictive expression network and analyzed it with random walk restart using 6 seed genes coding for transcription factors regulating xylem differentiation in poplar. Of the top ranked 200 transcripts, 119 transcripts were significant differentially expressed supporting these transcripts as potential candidates regulating resin canal formation. Analysis of single nuclei sequencing of shoot tips that contain differentiating resin canals, identified 10 clusters. One cluster was highly enriched in transcripts coding for 9 of the enzymes in the MEP pathway 3 prenyl synthetases, and 3 monoterpene synthases strongly suggesting that this cluster represents resin canal epithelial cells. We are mining the additional transcripts to create a trajectory analysis. In summary, we have identified > 10 novel genes that are strongly supported candidates for further analysis in breeding lines and for genetic engineering over- and under- expressing lines to increase wood terpene content to improve resistance to insect and fungal pathogens while simultaneously increasing terpene supplies for renewable chemicals and biofuels.
Peptide nucleic acid Hoogsteen strand linker design for major groove recognition of DNA thymine bases
Sequence-specific targeting of double-stranded DNA and non-coding RNA via triple-helix-forming peptide nucleic acids (PNAs) has attracted considerable attention in therapeutic, diagnostic and nanotechnological fields. An E-base (3-oxo-2,3-dihydropyridazine), attached to the polyamide backbone of a PNA Hoogsteen strand by a side-chain linker molecule, is typically used in the hydrogen bond recognition of the 4-oxo group of thymine and uracil nucleic acid bases in the major groove. We report on the application of quantum chemical computational methods, in conjunction with spatial constraints derived from the experimental structure of a homopyrimidine PNA·DNA-PNA hetero-triplex, to investigate the influence of linker flexibility on binding interactions of the E-base with thymine and uracil bases in geometry-optimised model systems. Hydrogen bond formation between the N2 E-base atom and target pyrimidine base 4-oxo groups in model systems containing a β-alanine linker (J Am Chem Soc 119:11116, 1997) was found to incur significant internal strain energy and the potential disruption of intra-stand aromatic base stacking interactions in an oligomeric context. Additionally, in geometry-optimised model systems containing a 3-trans olefin linker (Bioorg Med Chem Lett 14:1551, 2004) the E-base swung out away from the target pyrimidine bases into the solvent. These findings are in qualitative agreement with calorimetric measurements in hybridisation experiments at T–A and U–A inversion sites. In contrast, calculations on a novel 2-cis olefin linker design indicate that it could permit simultaneous E-base hydrogen bonding with the thymine 4-oxo group, circumvention and solvent screening of the thymine 5-methyl group, and maintenance of triplex intra-stand base stacking interactions.
Comparative study of two Saccharomyces cerevisiae strains with kinetic models at genome-scale
The parameterization of kinetic models requires measurement of fluxes and/or metabolite levels for a base strain and a few genetic perturbations thereof. Unlike stoichiometric models that are mostly invariant to the specific strain, it remains unclear whether kinetic models constructed for different strains of the same species have similar or significantly different kinetic parameters. This important question underpins the applicability range and prediction limits of kinetic reconstructions. To this end, herein we parameterize two separate large-scale kinetic models using K-FIT with genome-wide coverage corresponding to two distinct strains of Saccharomyces cerevisiae: CEN.PK 113-7D strain (model k-sacce306-CENPK), and growth-deficient BY4741 (isogenic to S288c; model k-sacce306-BY4741). The metabolic network for each model contains 306 reactions, 230 metabolites, and 119 substrate-level regulatory interactions. The two models (for CEN.PK and BY4741) recapitulate, within one standard deviation, 77% and 75% of the fitted dataset fluxes, respectively, determined by 13 C metabolic flux analysis for wild-type and eight single-gene knockout mutants of each strain. Strain-specific kinetic parameterization results indicate that key enzymes in the TCA cycle, glycolysis, and arginine and proline metabolism drive the metabolic differences between these two strains of S. cerevisiae. Our results suggest that although kinetic models cannot be readily used across strains as stoichiometric models, they can capture species-specific information through the kinetic parameterization process.
Experimental evolution reveals nitrate tolerance mechanisms in Desulfovibrio vulgaris
Elevated nitrate in the environment inhibits sulfate reduction by important microorganisms of sulfate-reducing bacteria (SRB). Several SRB may respire nitrate to survive under elevated nitrate, but how SRB that lack nitrate reductase survive to elevated nitrate remains elusive. To understand nitrate adaptation mechanisms, we evolved 12 populations of a model SRB (i.e., Desulfovibrio vulgaris Hildenborough, DvH) under elevated NaNO 3 for 1000 generations, analyzed growth and acquired mutations, and linked their genotypes with phenotypes. Nitrate-evolved (EN) populations significantly (p < 0.05) increased nitrate tolerance, and whole-genome resequencing identified 119 new mutations in 44 genes of 12 EN populations, among which six functional gene groups were discovered with high mutation frequencies at the population level. In this work, we observed a high frequency of nonsense or frameshift mutations in nitrosative stress response genes (NSR: DVU2543, DVU2547, and DVU2548), nitrogen regulatory protein C family genes (NRC: DVU2394-2396, DVU2402, and DVU2405), and nitrate cluster (DVU0246-0249 and DVU0251). Mutagenesis analysis confirmed that loss-of-functions of NRC and NSR increased nitrate tolerance. Also, functional gene groups involved in fatty acid synthesis, iron regulation, and two-component system (LytR/LytS) known to be responsive to multiple stresses, had a high frequency of missense mutations. Additionally, mutations in those gene groups could increase nitrate tolerance through regulating energy metabolism, barring entry of nitrate into cells, altering cell membrane characteristics, or conferring growth advantages at the stationary phase. This study advances our understanding of nitrate tolerance mechanisms and has important implications for linking genotypes with phenotypes in DvH.