Streamlining assays of glycosyltransferases activity using in vitro GT-array (i-GT-ray) platform: Application to family GT37 fucosyltransferases.

Numerous putative glycosyltransferases (GTs) have been identified using bioinformatic approaches. However, demonstrating the activity of these GTs remains a challenge. Here, we describe the development of a rapid in vitro GT-array screening platform for activity of GTs. GT-arrays are generated by cell-free in vitro protein synthesis and binding using microplates precoated with a N-terminal Halo- or a C-terminal GST-tagged GT-encoding plasmid DNA and a capture antibody. These arrays are then used for screening of transferase activities and the reactions are monitored by a luminescence GLO assay. The products formed by these reactions can be analyzed directly from the microplates by mass spectrometry. Using this platform, a total of 280 assays were performed to screen 22 putative fucosyltransferases (FUTs) from family GT37 (seven from Arabidopsis and 15 from rice) for activity toward five acceptors: non-fucosylated tamarind xyloglucan (TXyG), arabinotriose (Ara3), non-fucosylated rhamnogalacturonan I (RG-I), and RG-II from the mur1-1 Arabidopsis mutant, and the celery RG-II monomer lacking Arap and MeFuc of chain B and l-Gal of chain A. Our screen showed that AtFUT2, AtFUT5, and AtFUT10 have activity toward RG-I, while AtFUT8 was active on RG-II. Five rice OsFUTs have XyG-FUT activity and four rice OsFUTs have activity toward Ara3. None of the putative OsFUTs were active on the RG-I and RG-II. However, promiscuity toward acceptors was observed for several FUTs. These findings extend our knowledge of cell wall polysaccharide fucosylation in plants. We believe that in vitro GT-array platform provides a valuable tool for cell wall biochemistry and other research fields.

Specific protein interactions between rice members of the GT43 and GT47 families form various central cores of putative xylan synthase complexes.

Members of the glycosyltransferase (GT)43 and GT47 families have been associated with heteroxylan synthesis in both dicots and monocots and are thought to assemble into central cores of putative xylan synthase complexes (XSCs). Currently, it is unknown whether protein-protein interactions within these central cores are specific, how many such complexes exist, and whether these complexes are functionally redundant. Here, we used gene association network and co-expression approaches in rice to identify four OsGT43s and four OsGT47s that assemble into different GT43/GT47 complexes. Using two independent methods, we showed that (i) these GTs assemble into at least six unique complexes through specific protein-protein interactions and (ii) the proteins interact directly in vitro. Confocal microscopy showed that, when alone, all OsGT43s were retained in the endoplasmic reticulum (ER), while all OsGT47s were localized in the Golgi. co-expression of OsGT43s and OsGT47s displayed complexes that form in the ER but accumulate in Golgi. ER-to-Golgi trafficking appears to require interactions between OsGT43s and OsGT47s. Comparison of the central cores of the three putative rice OsXSCs to wheat, asparagus, and Arabidopsis XSCs, showed great variation in GT43/GT47 combinations, which makes the identification of orthologous central cores between grasses and dicots challenging. However, the emerging picture is that all central cores from these species seem to have at least one member of the IRX10/IRX10-L clade in the GT47 family in common, suggesting greater functional importance for this family in xylan synthesis. Our findings provide a new framework for future investigation of heteroxylan biosynthesis and function in monocots.

Barley Nodulin 26-like intrinsic protein permeates water, metalloids, saccharides, and ion pairs due to structural plasticity and diversification.

Aquaporins can facilitate the passive movement of water, small polar molecules, and some ions. Here, we examined solute selectivity for the barley Nodulin 26-like Intrinsic Protein (HvNIP2;1) embedded in liposomes and examined through stopped-flow light scattering spectrophotometry and Xenopus laevis oocyte swelling assays. We found that HvNIP2;1 permeates water, boric and germanic acids, sucrose, and lactose but not d-glucose or d-fructose. Other saccharides, such as neutral (d-mannose, d-galactose, d-xylose, d-mannoheptaose) and charged (N-acetyl d-glucosamine, d-glucosamine, d-glucuronic acid) aldoses, disaccharides (cellobiose, gentiobiose, trehalose), trisaccharide raffinose, and urea, glycerol, and acyclic polyols, were permeated to a much lower extent. We observed apparent permeation of hydrated KCl and MgSO4 ions, while CH3COONa and NaNO3 permeated at significantly lower rates. Our experiments with boric acid and sucrose revealed no apparent interaction between solutes when permeated together, and AgNO3 or H[AuCl4] blocked the permeation of all solutes. Docking of sucrose in HvNIP2;1 and spinach water-selective SoPIP2;1 aquaporins revealed the structural basis for sucrose permeation in HvNIP2;1 but not in SoPIP2;1, and defined key residues interacting with this permeant. In a biological context, sucrose transport could constitute a novel element of plant saccharide-transporting machinery. Phylogenomic analyses of 164 Viridiplantae and 2993 Archaean, bacterial, fungal, and Metazoan aquaporins rationalized solute poly-selectivity in NIP3 sub-clade entries and suggested that they diversified from other sub-clades to acquire a unique specificity of saccharide transporters. Solute specificity definition in NIP aquaporins could inspire developing plants for food production.