Acidothermus cellulolyticus E1 endoglucanase expressed in planta undergoes extensive hydroxyproline-O-glycosylation and exhibits enhanced impact on biomass digestibility.

KEY MESSAGE: E1 holoenzyme was extensively Hyp-O-glycosylated at the proline rich linker region in plants, which substantially increased the molecular size and improved the enzymatic digestibility of the biomass of transgenic plants. Thermophilic E1 endo-1,4-ß-glucanase derived from Acidothermus cellulolyticus has been frequently expressed in planta to reconstruct the plant cell wall to overcome biomass recalcitrance. However, the expressed holoenzyme exhibited a larger molecular size (~ 100 kDa) than the theoretical one (57 kDa), possibly due to posttranslational modifications in the recombinant enzyme within plant cells. This study investigates the glycosylation of the E1 holoenzyme expressed in tobacco plants and determines its impact on enzyme activity and biomass digestibility. The E1 holoenzyme, E1 catalytic domain (E1cd) and E1 linker (E1Lk) were each expressed in tobacco plants and suspension cells. The accumulation of holoenzyme was 2.0- to 2.3- times higher than that of E1cd. The proline-rich E1Lk region was extensively hydroxyproline-O-glycosylated with arabinogalactan polysaccharides. Compared with E1cd, the holoenzyme displayed a broader optimal temperature range (70 to 85 ºC). When grown in greenhouse, the expression of E1 holoenzyme induced notable phenotypic changes in plants, including delayed flowering and leaf variegation post-flowering. However, the final yield of plant biomass was not significantly affected. Finally, plant biomass engineering with E1 holoenzyme showed 1.7- to 1.8-fold higher saccharification efficiency than the E1cd lines and 2.4- to 2.7-fold higher than the wild-type lines, which was ascribed to the synergetic action of the E1Lk and cellulose binding module in reducing cell wall recalcitrance.

GST Pull-Down Assay to Study PIF4 Binding In Vitro.

The phytochrome-interacting factor 4 (PIF4) is a well-known transcription factor that plays a pivotal role in plant thermomorphogenesis, coordinating growth and development in response to temperature changes. As PIF4 functions by forming complexes with other proteins, determining its interacting partners is essential for understanding its diverse roles in plant thermal responses. The GST (glutathione-S-transferase) pull-down assay is a widely used biochemical technique that enables the investigation of protein-protein interactions in vitro. It is particularly useful for studying transient or weak interactions between proteins. In this chapter, we describe the GST pull-down approach to detect the interaction between PIF4 and a known or suspected interacting protein. We provide detailed step-by-step descriptions of the assay procedures, from the preparation of recombinant GST-PIF4 fusion protein to the binding and elution of interacting partners. Additionally, we provide guidelines for data interpretation, quantification, and statistical analysis to ensure robust and reliable results.

Intracellular trafficking and glycosylation of hydroxyproline-O-glycosylation module in tobacco BY-2 cells is dependent on medium composition and transcriptome analysis.

Expression of recombinant proteins in plant cells with a "designer" hydroxyproline (Hyp)-O-glycosylated peptide (HypGP), such as tandem repeats of a "Ser-Pro" motif, has been shown to boost the secreted protein yields. However, dramatic secretion and Hyp-O-glycosylation of HypGP-tagged proteins can only be achieved when the plant cells were grown in nitrogen-deficient SH medium. Only trace amounts of secreted fusion protein were detected in MS medium. This study aims to gain a deeper understanding of the possible mechanism underlying these results by examining the intracellular trafficking and Hyp-O-glycosylation of enhanced green fluorescent protein (EGFP) fused with a (SP)32 tag, consisting of 32 repeats of a "Ser-Pro" motif, in tobacco BY-2 cells. When cells were grown in MS medium, the (SP)32-EGFP formed protein body-like aggregate and was retained in the ER, without undergoing Hyp-O-glycosylation. In contrast, the fusion protein becomes fully Hyp-O-glycosylated, and then secreted in SH medium. Transcriptome analysis of the BY-2 cells grown in SH medium vs. MS medium revealed over 16,000 DEGs, with many upregulated DEGs associated with the microtubule-based movement, movement of subcellular component, and microtubule binding. These DEGs are presumably responsible for the enhanced ER-Golgi transport of HypGP-tagged proteins, enabling their glycosylation and secretion in SH medium.