Showing papers by "Timothy J. Tschaplinski published in 2023"

Mechanistic Insights into Cell-Free Gene Expression through an Integrated -Omics Analysis of Extract Processing Methods.

Abstract: Cell-free systems derived from crude cell extracts have developed into tools for gene expression, with applications in prototyping, biosensing, and protein production. Key to the development of these systems is optimization of cell extract preparation methods. However, the applied nature of these optimizations often limits investigation into the complex nature of the extracts themselves, which contain thousands of proteins and reaction networks with hundreds of metabolites. Here, we sought to uncover the black box of proteins and metabolites in Escherichia coli cell-free reactions based on different extract preparation methods. We assess changes in transcription and translation activity from σ70 promoters in extracts prepared with acetate or glutamate buffer and the common post-lysis processing steps of a runoff incubation and dialysis. We then utilize proteomic and metabolomic analyses to uncover potential mechanisms behind these changes in gene expression, highlighting the impact of cold shock-like proteins and the role of buffer composition.

Mechanism for Utilization of the Populus-Derived Metabolite Salicin by a Pseudomonas—Rahnella Co-Culture

Abstract: Pseudomonas fluorescens GM16 associates with Populus, a model plant in biofuel production. Populus releases abundant phenolic glycosides such as salicin, but P. fluorescens GM16 cannot utilize salicin, whereas Pseudomonas strains are known to utilize compounds similar to the aglycone moiety of salicin–salicyl alcohol. We propose that the association of Pseudomonas to Populus is mediated by another organism (such as Rahnella aquatilis OV744) that degrades the glucosyl group of salicin. In this study, we demonstrate that in the Rahnella–Pseudomonas salicin co-culture model, Rahnella grows by degrading salicin to glucose 6-phosphate and salicyl alcohol which is secreted out and is subsequently utilized by P. fluorescens GM16 for its growth. Using various quantitative approaches, we elucidate the individual pathways for salicin and salicyl alcohol metabolism present in Rahnella and Pseudomonas, respectively. Furthermore, we were able to establish that the salicyl alcohol cross-feeding interaction between the two strains on salicin medium is carried out through the combination of their respective individual pathways. The research presents one of the potential advantages of salicyl alcohol release by strains such as Rahnella, and how phenolic glycosides could be involved in attracting multiple types of bacteria into the Populus microbiome.

Variable lignin structure revealed in Populus leaves

Abstract: Lignin has long been a trait of interest, especially in bioenergy feedstocks such as Populus. While the stem lignin of Populus is well studied, foliar lignin has received significantly less consideration. To this end, leaves from 11 field grown, natural variant Populus trichocarpa genotypes were investigated by NMR, FTIR, and GC-MS. Five of these genotypes were sufficiently irrigated, and the other six genotypes were irrigated at a reduced rate (59% of the potential evapotranspiration for the site) to induce drought treatment. Analysis by HSQC NMR revealed highly variable lignin structure among the samples, especially for the syringyl/guaiacyl (S/G) ratio, which ranged from 0.52–11.9. Appreciable levels of a condensed syringyl lignin structure were observed in most samples. The same genotype subjected to different treatments exhibited similar levels of condensed syringyl lignin, suggesting this was not a response to stress. A cross peak of δC/δH 74.6/5.03, consistent with the erythro form of the β-O-4 linkage, was observed in genotypes where significant syringyl units were present. Principle component analysis revealed that FTIR absorbances associated with syringyl units (830 cm−1, 1317 cm−1) greatly contributed to variability between samples. Additionally, the ratio of 830/1230 cm−1 peak intensities were reasonably correlated (p-value < 0.05) with the S/G ratio determined by NMR. Analysis by GC-MS revealed significant variability of secondary metabolites such as tremuloidin, trichocarpin, and salicortin. Additionally, salicin derivatives were found to be well correlated with NMR results, which has been previously hypothesized. These results highlight previously unexplored nuance and variability associated with foliage tissue of poplar.

Novel candidate genes for lignin structure identified through genome-wide association study of naturally varying Populus trichocarpa

Abstract: Populus is a promising lignocellulosic feedstock for biofuels and bioproducts. However, the cell wall biopolymer lignin is a major barrier in conversion of biomass to biofuels. To investigate the variability and underlying genetic basis of the complex structure of lignin, a population of 409 three-year-old, naturally varying Populus trichocarpa genotypes were characterized by heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR). A subsequent genome-wide association study (GWAS) was conducted using approximately 8.3 million single nucleotide polymorphisms (SNPs), which identified 756 genes that were significantly associated (−log10(p-value)>6) with at least one lignin phenotype. Several promising candidate genes were identified, many of which have not previously been reported to be associated with lignin or cell wall biosynthesis. These results provide a resource for gaining insights into the molecular mechanisms of lignin biosynthesis and new targets for future genetic improvement in poplar.

Diel dynamics of multi-omics in elkhorn fern provides new insights into weak CAM photosynthesis.

Abstract: Crassulacean acid metabolism (CAM) has high water-use efficiency (WUE) and is widely recognized to have evolved from C3 photosynthesis. Different plant lineages have convergently evolved CAM, but the molecular mechanism that underlies C3-to-CAM evolution remains to be clarified. Platycerium bifurcatum (elkhorn fern) provides an opportunity to study the molecular changes underlying the transition from C3 to CAM photosynthesis because both modes of photosynthesis occur in this species, with sporotrophophyll leaves (SLs) and cover leaves (CLs) performing C3 and weak CAM photosynthesis, respectively. Here, we report that the physiological and biochemical attributes of CAM in weak CAM-performing CLs differed from those in strong CAM species. We investigated the diel dynamics of the metabolome, proteome, and transcriptome in these dimorphic leaves within the same genetic background and under identical environmental conditions. We found that multi-omic diel dynamics in P. bifurcatum exhibit both tissue and diel effects. Our analysis revealed temporal rewiring of biochemistry relevant to the energy-producing pathway (TCA cycle), CAM pathway, and stomatal movement in CLs compared with SLs. We also confirmed that PHOSPHOENOLPYRUVATE CARBOXYLASE KINASE (PPCK) exhibits convergence in gene expression among highly divergent CAM lineages. Gene regulatory network analysis identified candidate transcription factors regulating the CAM pathway and stomatal movement. Taken together, our results provide new insights into weak CAM photosynthesis and new avenues for CAM bioengineering.