Methodology for Constructing a Knowledgebase for Plant Gene Regulation Information.

The amount of biological data is growing at a rapid pace as many high-throughput omics technologies and data pipelines are developed. This is resulting in the growth of databases for DNA and protein sequences, gene expression, protein accumulation, structural, and localization information. The diversity and multi-omics nature of such bioinformatic data requires well-designed databases for flexible organization and presentation. Besides general-purpose online bioinformatic databases, users need narrowly focused online databases to quickly access a meaningful collection of related data for their research. Here, we describe the methodology used to implement a plant gene regulatory knowledgebase, with data, query, and tool features, as well as the ability to expand to accommodate future datasets. We exemplify this methodology for the GRASSIUS knowledgebase, but it is applicable to developing and updating similar plant gene regulatory knowledgebases. GRASSIUS organizes and presents gene regulatory data from grass species with a central focus on maize (Zea mays). The main class of data presented include not only the families of transcription factors (TFs) and co-regulators (CRs) but also protein-DNA interaction data, where available.

A structural explanation for the mechanism and specificity of plant branching enzymes I and IIb.

Branching enzymes (BEs) are essential in the biosynthesis of starch and glycogen and play critical roles in determining the fine structure of these polymers. The substrates of these BEs are long carbohydrate chains that interact with these enzymes via multiple binding sites on the enzyme's surface. By controlling the branched-chain length distribution, BEs can mediate the physiological properties of starch and glycogen moieties; however, the mechanism and structural determinants of this specificity remain mysterious. In this study, we identify a large dodecaose binding surface on rice BE I (BEI) that reaches from the outside of the active site to the active site of the enzyme. Mutagenesis activity assays confirm the importance of this binding site in enzyme catalysis, from which we conclude that it is likely the acceptor chain binding site. Comparison of the structures of BE from Cyanothece and BE1 from rice allowed us to model the location of the donor-binding site. We also identified two loops that likely interact with the donor chain and whose sequences diverge between plant BE1, which tends to transfer longer chains, and BEIIb, which transfers exclusively much shorter chains. When the sequences of these loops were swapped with the BEIIb sequence, rice BE1 also became a short-chain transferring enzyme, demonstrating the key role these loops play in specificity. Taken together, these results provide a more complete picture of the structure, selectivity, and activity of BEs.