Fig. 8: Heat response of gapc1gapc2 (DKO) complemented with GAPC2 variants.

a Co-immunoprecipitation of GAPC and NF-YC10. Purified GAPC2-6xHis or GAPC2mut’-6xHis was mixed with NF-YC10-6xHis-STREP. Immunoprecipitation (IP) was performed using an anti-STREP antibody, then immunoblotting was performed using an anti-6xHis antibody. Protein input is shown on the right. b GAPC expression levels. Total RNA was extracted from 10-day-old seedlings and quantitative RT-PCR was performed with GAPC2-specific primers. Values are average ± S.D. from 3 independent seedlings and shown as fold change to WT. Black dots represent individual data points. DKO, double knockout mutant of GAPC. Inset, immunoblotting of GAPC. Total proteins were extracted from 5-day-old seedlings and GAPC was probed with the anti-GAPC antibody. c Electrolyte leakage. Conductivity was measured with true leaves from 2-week-old plants immediately after heat treatment at 40 °C for 6 h. Electrolyte leakage was calculated and shown here as % conductivity of total ions (boiled sample). Values are average ± S.D. P values indicate significantly higher than WT determined by two-tailed student’s t test (n = 5 leaves from independent plants). Black dots represent individual data points. d Reactive oxygen species production. Rosette leaves from 3-week-old plants were incubated with NBT immediately after heat treatment at 40 °C for 6 h. NBT-formazan was quantified as absorbance at 700 nm and shown here as per tissue weight. Values are average ± S.D. P values indicate significant difference from WT determined by two-tailed student’s t test (n = 5 leaves from independent plants). Black dots represent individual data points. e Expression of heat-inducible genes. Total RNA was extracted from 10-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. P values indicate significantly lower than WT determined by two-tailed student’s t test (n = 3 independent seedlings). Black dots represent individual data points.