Summary: We developed a split luciferase complementation assay to study protein–protein interactions in Arabidopsis protoplasts. In this assay, the N‐ and C‐terminal fragments of Renilla reniforms luciferase are translationally fused to bait and prey proteins, respectively. When the proteins interact, split luciferase becomes activated and emits luminescence that can be measured by a microplate luminometer. Split luciferase activity was measured by first transforming protoplasts with a DNA vector in a 96‐well plate. DNA vector expressing both bait and prey genes was constructed through two independent in vitro DNA recombinant reactions, Gateway and Cre‐loxP. As proof of concept, we detected the protein–protein interactions between the nuclear histones 2A and 2B, as well as between membrane proteins SYP (syntaxin of plant) 51 and SYP61, in Arabidopsis protoplasts.

The cyclin-dependent kinase inhibitor SIAMESE (SIM) targets both CDKA;1 and CDKB1 complexes, leading to establishment of endoreplication, and SIM interacts with

A systems model that describes vesicle trafficking during pollen tube growth in Arabidopsis (Arabidopsis thaliana) was constructed. The model is composed of ordinary differential equations that connect the molecular functions of genes expressed in pollen. The current model requires soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) and small GTPases, Arf or Rab, to reasonably predict tube growth as a function of time. Tube growth depends on vesicle trafficking that transports phospholipid and pectin to the tube tip. The vesicle trafficking genes identified by analyzing publicly available transcriptome data comprised 328 genes. Fourteen of them are up-regulated by the gibberellin signaling pathway during pollen development, which includes the SNARE genes SYP124 and SYP125 and the Rab GTPase gene RABA4D. The model results adequately fit the pollen tube growth of both previously reported wild-type and raba4d knockout lines. Furthermore, the difference of pollen tube growth in syp124/syp125 single and double mutations was quantitatively predicted based on the model analysis. In general, a systems model approach to vesicle trafficking arguably demonstrated the importance of the functional connections in pollen tube growth and can help guide future research directions.

Background: The rice interactome, in which a network of protein-protein interactions has been elucidated in rice, is a useful resource to identify functional modules of rice signal transduction pathways. Protein-protein interactions occur in cells in two ways, constitutive and regulative. While a yeast-based high-throughput method has been widely used to identify the constitutive interactions, a method to detect the regulated interactions is rarely developed for a large-scale analysis.Results: A split luciferase complementation assay was applied to detect the regulated interactions in rice. A transformation method of rice protoplasts in a 96-well plate was first established for a large-scale analysis. In addition, an antibody that specifically recognizes a carboxyl-terminal fragment ofRenillaluciferase was newly developed. A pair of antibodies that recognize amino- and carboxyl- terminal fragments ofRenillaluciferase, respectively, was then used to monitor quality and quantity of interacting recombinant-proteins accumulated in the cells. For a proof-of-concept, the method was applied to detect the gibberellin-dependent interaction between GIBBERELLIN INSENSITIVE DWARF1 and SLENDER RICE 1.Conclusions: A method to detect regulated protein-protein interactions was developed towards establishment of the rice interactome.

The abscisic acid (ABA) signaling pathway is the key defense mechanism against drought stress in plants. In the pathway, signal transduction among four core proteins, pyrabactin resistance (PYR), protein phosphatase 2C (PP2C), sucrose-non-fermenting-1-related protein kinase 2 (SnRK2), and ABRE binding factor (ABF) leads to altered gene expression kinetics that is driven by an ABA-responsive element (ABRE). A most recent and comprehensive study provided data suggesting that ABA alters the expression kinetics in over 6,500 genes through the ABF-ABRE associations in Arabidopsis. Of these genes, termed ABA gene regulatory network (GRN), over 50% contain a single ABRE within 4 kb of the gene body, despite previous findings suggesting that a single copy of ABRE is not sufficient to drive the gene expression. To understand the expression system of the ABA GRN by the single ABRE, a dynamic model of the gene expression for the desiccation 29A (RD29A) gene was constructed with ordinary differential equations. Parameter values of molecular-molecular interactions and enzymatic reactions in the model were implemented from the data obtained by previously conducted in vitro experiments. On the other hand, parameter values of gene expression and translation were determined by comparing the kinetics of gene expression in the model to the expression kinetics of RD29A in real plants. The optimized model recapitulated the trend of gene expression kinetics of RD29A in ABA dose–response that were previously investigated. Further analysis of the model suggested that a single ABRE controls the time scale and dynamic range of the ABA-dependent gene expression through the PP2C feedback regulation even though an additional cis-element is required to drive the expression. The model construed in this study underpins the importance of a single ABRE in the ABA GRN.