Fig. 7: Effects of GAPC mutation on heat response.

a GAPC2 amino acid sequence. Colored are the lysine residues required for HsGAPDH nuclear localization (K121 and K231 in red, K255 in green) and found to be acetylated in E. coli (K130, K219, and K223 in blue, K255 in green). The lysines mutated in this study are underlined. b Immununoblotting of GAPC in nuclear fraction. Five-day-old transgenic seedlings overexpressing the indicated GAPC-Flag were untreated (Control) or treated at 40 °C for 6 h. Nuclei were isolated and GAPC was probed with an anti-Flag antibody by immunoblotting. Histone H3 was used as a nuclear marker. GAPCmut, GAPC with K121A/K231A. c Fluorescence images of Arabidopsis leaf cells. Five-day-old seedlings overexpressing the indicated GAPC-GFP were treated at 40 °C for 6 h and observed under a confocal microscope. Arrows indicate the nucleus. Scale bars = 10 µm. d The number of cells with nuclear GAPC. Plants were treated and observed as in c. Cells with clear fluorescence in the nucleus were counted and shown here as % of total cells counted. Values are average ± S.D. from 5 leaves independently treated. Black dots represent individual data points. e Quantitative measurement of heat response. Plants were untreated (Control) or treated at 40 °C for 6 h and measured for seedling survival rate. Values are average ± S.D. P values indicate significant difference from WT determined by two-tailed student’s t test (n = 3 independent groups of >30 seedlings). Black dots represent individual data points. f Expression of heat-inducible genes. Total RNA was extracted from 5-day-old seedlings treated at 37 °C for 5 h and quantitative RT-PCR was performed with gene-specific primers. Values are average ± S.D. and shown as fold change to WT (dashed line). P values indicate significant difference from WT determined by two-tailed student’s t test (n = 3 independent groups of >10 seedlings). Black dots represent individual data points.