Identifying new lead structures to enhance tolerance towards drought stress via high-throughput screening giving crops a quantum of solace.

Novel synthetic lead structures interacting with RCAR/(PYR/PYL) receptor proteins were identified based on the results of a high-throughput screening campaign of a large compound library followed by focused SAR studies of the three most promising hit clusters. Whilst indolinylmethyl sulfonamides 8y,z and phenylsulfonyl ethylenediamines 9y,z showed strong affinities for RCAR/ (PYR/PYL) receptor proteins in wheat, thiotriazolyl acetamides 7f,s exhibited promising efficacy against drought stress in vivo (wheat, corn and canola) combined with confirmed target interaction in wheat and arabidopsis thaliana. Remarkably, binding affinities of several representatives of 8 and 9 were on the same level or even better than the essential plant hormone abscisic acid (ABA).

Glycome and Proteome Components of Golgi Membranes Are Common between Two Angiosperms with Distinct Cell-Wall Structures.

The plant endoplasmic reticulum-Golgi apparatus is the site of synthesis, assembly, and trafficking of all noncellulosic polysaccharides, proteoglycans, and proteins destined for the cell wall. As grass species make cell walls distinct from those of dicots and noncommelinid monocots, it has been assumed that the differences in cell-wall composition stem from differences in biosynthetic capacities of their respective Golgi. However, immunosorbence-based screens and carbohydrate linkage analysis of polysaccharides in Golgi membranes, enriched by flotation centrifugation from etiolated coleoptiles of maize (Zea mays) and leaves of Arabidopsis (Arabidopsis thaliana), showed that arabinogalactan-proteins and arabinans represent substantial portions of the Golgi-resident polysaccharides not typically found in high abundance in cell walls of either species. Further, hemicelluloses accumulated in Golgi at levels that contrasted with those found in their respective cell walls, with xyloglucans enriched in maize Golgi, and xylans enriched in Arabidopsis. Consistent with this finding, maize Golgi membranes isolated by flotation centrifugation and enriched further by free-flow electrophoresis, yielded >200 proteins known to function in the biosynthesis and metabolism of cell-wall polysaccharides common to all angiosperms, and not just those specific to cell-wall type. We propose that the distinctive compositions of grass primary cell walls compared with other angiosperms result from differential gating or metabolism of secreted polysaccharides post-Golgi by an as-yet unknown mechanism, and not necessarily by differential expression of genes encoding specific synthase complexes.

Free-Flow Electrophoresis of Plasma Membrane Vesicles Enriched by Two-Phase Partitioning Enhances the Quality of the Proteome from Arabidopsis Seedlings.

The plant plasma membrane is the interface between the cell and its environment undertaking a range of important functions related to transport, signaling, cell wall biosynthesis, and secretion. Multiple proteomic studies have attempted to capture the diversity of proteins in the plasma membrane using biochemical fractionation techniques. In this study, two-phase partitioning was combined with free-flow electrophoresis to produce a population of highly purified plasma membrane vesicles that were subsequently characterized by tandem mass spectroscopy. This combined high-quality plasma membrane isolation technique produced a reproducible proteomic library of over 1000 proteins with an extended dynamic range including plasma membrane-associated proteins. The approach enabled the detection of a number of putative plasma membrane proteins not previously identified by other studies, including peripheral membrane proteins. Utilizing multiple data sources, we developed a PM-confidence score to provide a value indicating association to the plasma membrane. This study highlights over 700 proteins that, while seemingly abundant at the plasma membrane, are mostly unstudied. To validate this data set, we selected 14 candidates and transiently localized 13 to the plasma membrane using a fluorescent tag. Given the importance of the plasma membrane, this data set provides a valuable tool to further investigate important proteins. The mass spectrometry data are available via ProteomeXchange, identifier PXD001795.

Standard flow liquid chromatography for shotgun proteomics in bioenergy research.

Over the past 10 years, the bioenergy field has realized significant achievements that have encouraged many follow on efforts centered on biosynthetic production of fuel-like compounds. Key to the success of these efforts has been transformational developments in feedstock characterization and metabolic engineering of biofuel-producing microbes. Lagging far behind these advancements are analytical methods to characterize and quantify systems of interest to the bioenergy field. In particular, the utilization of proteomics, while valuable for identifying novel enzymes and diagnosing problems associated with biofuel-producing microbes, is limited by a lack of robustness and limited throughput. Nano-flow liquid chromatography coupled to high-mass accuracy, high-resolution mass spectrometers has become the dominant approach for the analysis of complex proteomic samples, yet such assays still require dedicated experts for data acquisition, analysis, and instrument upkeep. The recent adoption of standard flow chromatography (ca. 0.5 mL/min) for targeted proteomics has highlighted the robust nature and increased throughput of this approach for sample analysis. Consequently, we assessed the applicability of standard flow liquid chromatography for shotgun proteomics using samples from Escherichia coli and Arabidopsis thaliana, organisms commonly used as model systems for lignocellulosic biofuels research. Employing 120 min gradients with standard flow chromatography, we were able to routinely identify nearly 800 proteins from E. coli samples; while for samples from Arabidopsis, over 1,000 proteins could be reliably identified. An examination of identified peptides indicated that the method was suitable for reproducible applications in shotgun proteomics. Standard flow liquid chromatography for shotgun proteomics provides a robust approach for the analysis of complex samples. To the best of our knowledge, this study represents the first attempt to validate the standard flow approach for shotgun proteomics.

The plant glycosyltransferase clone collection for functional genomics.

The glycosyltransferases (GTs) are an important and functionally diverse family of enzymes involved in glycan and glycoside biosynthesis. Plants have evolved large families of GTs which undertake the array of glycosylation reactions that occur during plant development and growth. Based on the Carbohydrate-Active enZymes (CAZy) database, the genome of the reference plant Arabidopsis thaliana codes for over 450 GTs, while the rice genome (Oryza sativa) contains over 600 members. Collectively, GTs from these reference plants can be classified into over 40 distinct GT families. Although these enzymes are involved in many important plant specific processes such as cell-wall and secondary metabolite biosynthesis, few have been functionally characterized. We have sought to develop a plant GTs clone resource that will enable functional genomic approaches to be undertaken by the plant research community. In total, 403 (88%) of CAZy defined Arabidopsis GTs have been cloned, while 96 (15%) of the GTs coded by rice have been cloned. The collection resulted in the update of a number of Arabidopsis GT gene models. The clones represent full-length coding sequences without termination codons and are Gateway® compatible. To demonstrate the utility of this JBEI GT Collection, a set of efficient particle bombardment plasmids (pBullet) was also constructed with markers for the endomembrane. The utility of the pBullet collection was demonstrated by localizing all members of the Arabidopsis GT14 family to the Golgi apparatus or the endoplasmic reticulum (ER). Updates to these resources are available at the JBEI GT Collection website http://www.addgene.org/.