Elucidating the Temporal Patterns of Gene Expression in the Inferred Regulatory Interactions of GmCOL1b in Glycine max

Abstract

The CONSTANS ( CO ) gene in Arabidopsis thaliana has a central role in photoperiodic regulation of flowering. However, the roles of CO genes in mediating flowering in soybeans ( Glycine max ) remain uncertain. We previously inferred regulatory interactions of a soybean CO homolog, GmCOL1b , using in-house RNA-seq data and the network inference algorithm package CausNet. Here, we identify potential GmCOL1b downstream genes and experimentally verify them by expressing GmCOL1b in soybean protoplast cells. Temporal expression patterns of these genes indicate the regulatory effects of GmCOL1b on the expression of the circadian clock genes GmLCL1 and GmLCL4 and the flowering regulator GmTEM1a .

Figure 1. Expression of GFP-GmCOL1b and inferred downstream genes in GFP-GmCOL1b transfected protoplasts.

A) Flowchart of experimental steps used in this study. B) Representative images of non-transfected and GFP-GmCOL1b transfected protoplasts in bright field (BF) and fluorescent light at a wavelength of 523nm (514-527nm range) showing cell viability and GFP expression. To determine cell viability, protoplasts were stained with FDA at the time of harvest. (C-F) Relative expression of GmCOL1b (C) and inferred GmCOL1b target genes: GmTEM1a (D), GmLCL1 (E), and GmLCL4 (F) in non-transfected negative control protoplasts (blue) and GFP-GmCOL1b transfected protoplasts (green) at Zeitgeber time points ZT4-ZT24 by RT-qPCR in two biological samples with three technical replications. Normalized expression levels were calculated as 2 ^-∆Ct against the housekeeping gene GmPBB2 as described previously (Livak & Schmittgen, 2001; Wu et al., 2014). Relative gene expression levels were calculated for each graph using the ZT4 expression level in the Negative Control cells as 1. Error bars indicate standard error with 2 biological replicates.