Figure 3

The transient expression of GmNAC6 in leaf soybean protoplasts induces cell death. Transient expression of GmNAC6 in protoplasts. Soybean protoplasts were electroporated with the 35S::YFP-NAC6 construct or the empty vector, and the expression of GmNAC6 and YFP-GmNAC6 was monitored by quantitative RT-PCR 36-h after electroporation. Values of expression were calculated using the 2^-ΔCt method and helicase as endogenous control. Values represent the mean ± SD of three replicates. B. Caspase-3-like activity. Total protein was extracted from GmNAC6-electroporated protoplasts after 36 h, and caspase 3-like activities were monitored with a DEVD-pNA substrate in the absence and presence of a specific inhibitor. Values represent the mean ± SD of three replicates. C. DNA fragmentation promoted by GmNAC6 expression. Cells were sampled 36-h post-electroporation of soybean protoplasts with empty vector or GmNAC6 expression cassette, submitted to TUNEL labeling and examined by confocal microscopy. The cells were also counterstained with propidium iodide (PI) and examined for red fluorescence at 632 nm. Arrows indicate some nuclei. Merged is an overlay of the fluorescent image of TUNEL labeling with PI staining cells to facilitate the identification of TUNEL-positive nuclei. As a positive control, untransfected cells were also treated with DNase.