Figure 6.

Functional complementation of phytaspase deficiency in methyl viologen (MV)-treated tobacco leaves. (A) Heterologous expression of the wild-type rice phytaspase gene (Rp-wt) in the phytaspase-silenced transgenic tobacco leaves, 24 h before stress treatments led to restoration of the wild-type PCD phenotype as exemplified for MV stress, whereas S⁵³⁵A mutant of phytaspase (KD + Rp-S⁵³⁵A) did not affect viability of leaf discs. Data are shown for three independent RNAi silencing lines (KD1-1, KD2-1 and KD3-1). MV at the concentration of 10 μM induced PCD in wild type (wt), but not in phytaspase RNAi (KD) plants. Photographs were taken 48 h after treatment. (B) Accumulation of H₂O₂ induced by MV (versus untreated control) was suppressed in phytaspase-silenced leaf discs compared with wild-type leaves and was restored by Agrobacterium-mediated transient expression of wild-type rice phytaspase (Rp-wt), whereas it was unaffected by S⁵³⁵A mutant of phytaspase (Rp-S⁵³⁵A). H₂O₂ production was determined 24 h after treatment. Data are means±s.d. from three independent experiments with three replicates in each. Data (including photographs) were typical of all generated KD and OE transgenic lines, respectively, tested in, at least, three experiments with three replicates in each.