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. 2019 Dec 12;295(3):783–799. doi: 10.1074/jbc.RA119.010779

Figure 3.

Figure 3.

AtPIMT1 and AtPIMT2 RNAi lines are hypersensitive to heat and oxidative stresses. a, phenotype of AtPIMT1 RNAi, AtPIMT2 RNAi, and WT seedlings (10-day-old) under control, heat, and oxidative stress conditions. Three representative independent lines of AtPIMT1 RNAi (lines 3, 5, and 6) and AtPIMT2 RNAi (lines 2, 5, and 6) are shown here. Five-day-old Arabidopsis seedlings were transferred to ½MS plates subjected to oxidative (1 μm paraquat) and heat stress (37 °C). The phenotype of WT and transgenic seedlings was monitored and photographed. Transgenic lines with variable phenotypes were purposely shown here. b, biomass accumulation of AtPIMT1 RNAi, AtPIMT2 RNAi, and WT seedlings under control, heat, and oxidative stress conditions. Fresh weight of seedlings was measured 3 days post-treatments. The results are presented as total biomass of 20 seedlings. The values represent mean ± S.E. (n = 3, 20 seedlings each). c, chlorophyll content of AtPIMT1 RNAi, AtPIMT2 RNAi, and WT seedlings grown under control, oxidative, and heat stress. d, root elongation of AtPIMT1 RNAi, AtPIMT2 RNAi, and WT seedlings grown under control, heat, and oxidative stress conditions. Elongation of root was determined by measuring root growth during 24-h post-stress treatments using ImageJ software. The values are shown in box plots (n = 30). e, growth pattern of soil-grown Arabidopsis mature plants of AtPIMT1 RNAi, AtPIMT2 RNAi, and WT under control, heat, and oxidative stress conditions. Four-week-old WT and transgenic plants were subjected to oxidative stress (10 μm paraquat and 0.05% Triton X-100) and heat stress (37 °C). Growth parameters are as follows: plant height (f), no. of siliques (g); and rosette diameter (h) were measured of AtPIMT1 RNAi, AtPIMT2 RNAi, and WT plants grown under control, heat, and oxidative stress conditions. The box plots represent values obtained from 10 independent plants per line. Significant differences (α = 0.01) are denoted by the different letters. (The RNAi lines died at rosette stage after paraquat treatment. Therefore, plant height and no. of siliques in response to oxidative stress could not be measured). i and j, quantitation of PIMT activity (i) and isoAsp accumulation (j) in proteins extracted from seedlings grown under control, heat, and oxidative stress conditions. Fifty micrograms of crude protein were used for isoAsp estimation and PIMT activity. Data are means ± S.E. of four biological replicates. Significant differences (α = 0.01) are denoted by the different letters. Control is shown in lowercase letters, oxidative stress in uppercase letters, and heat stress in lowercase letters with a prime. Scale bar = 1 cm for a and e.