Figure 5.
Involvement of ROS and the Accumulation of Defense Markers during Cell Death.
(A) ROS production. Protoplasts isolated from leaves of TRV or TRV:Hxk1 VIGS lines were incubated in 2 μM H2DCFDA for 90 s. H2DCFDA is a ROS indicator that becomes fluorescent when oxidized by ROS within the cell. The fluorescence of protoplasts from the TRV and TRV:Hxk1 VIGS lines was quantified by pixel intensity. Data points represent means ± sd of 13 to 16 individual protoplasts. PIV, pixel intensity value.
(B) Lignin deposition. Leaf samples were incubated in a phloroglucinol-HCl solution for 2 min. Lignin staining appears pink.
(C) Relative ion leakage. Data points are indicated as relative values (TRV = 100%). Means ± sd of three experiments per line are indicated.
(D) Semiquantitative RT-PCR analysis to examine transcript levels of cell death– and defense–related genes. Total RNA was extracted from the fourth leaf above the infiltrated leaves from the TRV and TRV:Hxk1 lines. As a control for RNA amounts, actin mRNA levels were examined.