Figure 5.

Ectopic expression of PrpS and PrsS in Arabidopsis triggers cell death in whole seedlings in an S-specific manner. A and B, Root growth of XVE-PrsS₁/PrpS₁ seedlings was inhibited after estradiol induction in an S-specific manner. A, Four-day-old seedlings were transferred to new medium containing 10 µm estradiol. Images were taken 48 h after treatment. White dashed lines indicate the positions of the root tips at the time of transfer. Estradiol-induced expression of PrsS₁ resulted in the death of the whole seedling (top, center), whereas no obvious effect was observed when PrsS₃ was expressed (top, right). B, Quantification of root length at 24 and 48 h after estradiol induction reveals the inhibition of root growth in XVE-PrsS₁/PrpS₁ lines upon transfer to estradiol plates, whereas the growth of roots of XVE-PrsS₃/PrpS₁ and pUBQ10::PrpS₁ seedlings was not affected (means ± sd; n = 20 seedlings). C, Estradiol induction resulted in nuclear disintegration and cell death of XVE-PrsS₁/PrpS₁ seedlings. NLS-YC3.6 was used to monitor the nuclear integrity after estradiol induction over time. Confocal images of merged eCFP (green) and cpVENUS (red) channels are shown. The yellow signal (green-red overlap) shows intact nuclei; extensive nuclear disintegration (loss of yellow signal) was observed as early as 5 h after estradiol induction and was almost complete by 11 h. The fluorescence signal was so weak at 5 h that the confocal laser power was increased from 1.5% (0 and 3 h) to 3.5% (5, 7, 11, and 24 h) to allow visualization of the seedling. NLS-YC3.6 monitors [Ca²⁺]_nuc and reveals that increases (red signal; indicated by white arrowheads) could be observed 3 h after induction. Bar = 100 µm. D, PI staining of a representative root at 24 h after estradiol induction reveals that virtually all the cells are dead (white signal). Bar = 100 µm.