Characterization of Transcription Regulatory Domains of OsMADS29: Identification of Proximal Auxin-Responsive Domains and a Strong Distal Negative Element.

OsMADS29 (M29) is a seed-specific MADS-box transcription factor involved in programmed cell death of nucellar tissue and maintaining auxin:cytokinin homeostasis. It affects embryo and endosperm development and starch filling during seed development in rice. Its expression seems to be tightly regulated by developmental, spatial, and temporal cues; however, cis- and trans-regulatory factors that affect its expression are largely unknown. In silico analysis of the 1.7 kb upstream regulatory region (URR) consisting of 1,290 bp promoter and 425 bp 5'-UTR regions revealed several auxin-responsive and seed-specific cis-regulatory elements distributed across the URR. In this study, the analysis of four URR deletions fused to a downstream ß-glucuronidase (GUS) reporter in transgenic rice has revealed the presence of several proximal positive elements and a strong distal negative element (NE). The promoter regions containing auxin-responsive elements responded positively to the exogenous application of auxins to transgenic seedlings. The proximal positive elements are capable of driving reporter expression in both vegetative and reproductive tissues. In contrast, the NE strongly suppresses reporter gene expression in both vegetative and reproductive tissues. In a transient onion peel assay system, the NE could reduce the efficacy of a 2x CaMV 35S promoter by ∼90%. Our results indicate the existence of a complex array of positive and negative regulatory regions along with auxin-responsive elements guiding the development-dependent and spatial expression of M29.

Determination of Tripartite Interaction between Two Monomers of a MADS-box Transcription Factor and a Calcium Sensor Protein by BiFC-FRET-FLIM Assay.

Protein-protein interactions are an integral part of all biological processes in the cells as they play a crucial role in regulating, maintaining, and amending cellular functions. These interactions are involved in a wide range of phenomena such as signal transduction, pathogen response, cell-cell interactions, metabolic and developmental processes. In the case of transcription factors, these interactions may lead to oligomerization of subunits, sequestering in specific subcellular contexts such as the nucleus, cytoplasm, etc., which, in turn, might have a more profound effect on the expression of the downstream genes. Here, we demonstrate a methodology to visualize in vivo tripartite interaction using Bimolecular Fluorescence Complementation (BiFC) based Förster Resonance Energy Transfer (FRET) involving Fluorescence Lifetime Imaging (FLIM). Two of the proteins selected for this demonstration interact as BiFC partners, and their reconstituted fluorescence activity is used to assay FRET-FLIM with the third partner. Four to five-week-old growth-chamber-grown Nicotiana benthamiana plants have been used as the model plant system for this demonstration.