Plant cell wall glycosyltransferases: High-throughput recombinant expression screening and general requirements for these challenging enzymes.

Molecular characterization of plant cell wall glycosyltransferases is a critical step towards understanding the biosynthesis of the complex plant cell wall, and ultimately for efficient engineering of biofuel and agricultural crops. The majority of these enzymes have proven very difficult to obtain in the needed amount and purity for such molecular studies, and recombinant cell wall glycosyltransferase production efforts have largely failed. A daunting number of strategies can be employed to overcome this challenge, including optimization of DNA and protein sequences, choice of expression organism, expression conditions, co-expression partners, purification methods, and optimization of protein solubility and stability. Hence researchers are presented with thousands of potential conditions to test. Ultimately, the subset of conditions that will be sampled depends on practical considerations and prior knowledge of the enzyme(s) being studied. We have developed a rational approach to this process. We devise a pipeline comprising in silico selection of targets and construct design, and high-throughput expression screening, target enrichment, and hit identification. We have applied this pipeline to a test set of Arabidopsis thaliana cell wall glycosyltransferases known to be challenging to obtain in soluble form, as well as to a library of cell wall glycosyltransferases from other plants including agricultural and biofuel crops. The screening results suggest that recombinant cell wall glycosyltransferases in general have a very low soluble:insoluble ratio in lysates from heterologous expression cultures, and that co-expression of chaperones as well as lysis buffer optimization can increase this ratio. We have applied the identified preferred conditions to Reversibly Glycosylated Polypeptide 1 from Arabidopsis thaliana, and processed this enzyme to near-purity in unprecedented milligram amounts. The obtained preparation of Reversibly Glycosylated Polypeptide 1 has the expected arabinopyranose mutase and autoglycosylation activities.

X-ray diffraction analysis and in vitro characterization of the UAM2 protein from Oryza sativa.

The role of seemingly non-enzymatic proteins in complexes interconverting UDP-arabinopyranose and UDP-arabinofuranose (UDP-arabinosemutases; UAMs) in the plant cytosol remains unknown. To shed light on their function, crystallographic and functional studies of the seemingly non-enzymatic UAM2 protein from Oryza sativa (OsUAM2) were undertaken. Here, X-ray diffraction data are reported, as well as analysis of the oligomeric state in the crystal and in solution. OsUAM2 crystallizes readily but forms highly radiation-sensitive crystals with limited diffraction power, requiring careful low-dose vector data acquisition. Using size-exclusion chromatography, it is shown that the protein is monomeric in solution. Finally, limited proteolysis was employed to demonstrate DTT-enhanced proteolytic digestion, indicating the existence of at least one intramolecular disulfide bridge or, alternatively, a requirement for a structural metal ion.

Transgenic expression of fungal accessory hemicellulases in Arabidopsis thaliana triggers transcriptional patterns related to biotic stress and defense response.

The plant cell wall is an abundant and renewable resource for lignocellulosic applications such as the production of biofuel. Due to structural and compositional complexities, the plant cell wall is, however, recalcitrant to hydrolysis and extraction of platform sugars. A cell wall engineering strategy to reduce this recalcitrance makes use of microbial cell wall modifying enzymes that are expressed directly in plants themselves. Previously, we constructed transgenic Arabidopsis thaliana constitutively expressing the fungal hemicellulases: Phanerochaete carnosa glucurnoyl esterase (PcGCE) and Aspergillus nidulans α-arabinofuranosidase (AnAF54). While the PcGCE lines demonstrated improved xylan extractability, they also displayed chlorotic leaves leading to the hypothesis that expression of such enzymes in planta resulted in plant stress. The objective of this study is to investigate the impact of transgenic expression of the aforementioned microbial hemicellulases in planta on the host arabidopsis. More specifically, we investigated transcriptome profiles by short read high throughput sequencing (RNAseq) from developmentally distinct parts of the plant stem. When compared to non-transformed wild-type plants, a subset of genes was identified that showed differential transcript abundance in all transgenic lines and tissues investigated. Intriguingly, this core set of genes was significantly enriched for those involved in plant defense and biotic stress responses. While stress and defense-related genes showed increased transcript abundance in the transgenic plants regardless of tissue or genotype, genes involved in photosynthesis (light harvesting) were decreased in their transcript abundance potentially reflecting wide-spread effects of heterologous microbial transgene expression and the maintenance of plant homeostasis. Additionally, an increase in transcript abundance for genes involved in salicylic acid signaling further substantiates our finding that transgenic expression of microbial cell wall modifying enzymes induces transcriptome responses similar to those observed in defense responses.

Loss of Inositol Phosphorylceramide Sphingolipid Mannosylation Induces Plant Immune Responses and Reduces Cellulose Content in Arabidopsis.

Glycosylinositol phosphorylceramides (GIPCs) are a class of glycosylated sphingolipids found in plants, fungi, and protozoa. These lipids are abundant in the plant plasma membrane, forming ∼25% of total plasma membrane lipids. Little is known about the function of the glycosylated headgroup, but two recent studies have indicated that they play a key role in plant signaling and defense. Here, we show that a member of glycosyltransferase family 64, previously named ECTOPICALLY PARTING CELLS1, is likely a Golgi-localized GIPC-specific mannosyl-transferase, which we renamed GIPC MANNOSYL-TRANSFERASE1 (GMT1). Sphingolipid analysis revealed that the Arabidopsis thaliana gmt1 mutant almost completely lacks mannose-carrying GIPCs. Heterologous expression of GMT1 in Saccharomyces cerevisiae and tobacco (Nicotiana tabacum) cv Bright Yellow 2 resulted in the production of non-native mannosylated GIPCs. gmt1 displays a severe dwarfed phenotype and a constitutive hypersensitive response characterized by elevated salicylic acid and hydrogen peroxide levels, similar to that we previously reported for the Golgi-localized, GIPC-specific, GDP-Man transporter GONST1 (Mortimer et al., 2013). Unexpectedly, we show that gmt1 cell walls have a reduction in cellulose content, although other matrix polysaccharides are unchanged.

Towards practical time-of-flight secondary ion mass spectrometry lignocellulolytic enzyme assays.

BACKGROUND: Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) is a surface sensitive mass spectrometry technique with potential strengths as a method for detecting enzymatic activity on solid materials. In particular, ToF-SIMS has been applied to detect the enzymatic degradation of woody lignocellulose. Proof-of-principle experiments previously demonstrated the detection of both lignin-degrading and cellulose-degrading enzymes on solvent-extracted hardwood and softwood. However, these preliminary experiments suffered from low sample throughput and were restricted to samples which had been solvent-extracted in order to minimize the potential for mass interferences between low molecular weight extractive compounds and polymeric lignocellulose components. RESULTS: The present work introduces a new, higher-throughput method for processing powdered wood samples for ToF-SIMS, meanwhile exploring likely sources of sample contamination. Multivariate analysis (MVA) including Principal Component Analysis (PCA) and Multivariate Curve Resolution (MCR) was regularly used to check for sample contamination as well as to detect extractives and enzyme activity. New data also demonstrates successful ToF-SIMS analysis of unextracted samples, placing an emphasis on identifying the low-mass secondary ion peaks related to extractives, revealing how extractives change previously established peak ratios used to describe enzyme activity, and elucidating peak intensity patterns for better detection of cellulase activity in the presence of extractives. The sensitivity of ToF-SIMS to a range of cellulase doses is also shown, along with preliminary experiments augmenting the cellulase cocktail with other proteins. CONCLUSIONS: These new procedures increase the throughput of sample preparation for ToF-SIMS analysis of lignocellulose and expand the applications of the method to include unextracted lignocellulose. These are important steps towards the practical use of ToF-SIMS as a tool to screen for changes in plant composition, whether the transformation of the lignocellulose is achieved through enzyme application, plant mutagenesis, or other treatments.