Fig. 4

Improved thermotolerance through AtNIA2 overexpression in a cngc6 background. (a) RT-qPCR analysis of AtCNGC6 and AtNIA2 transcription in wild-type, cngc6, cngc6/35S::NIA2–1, and cngc6/35S::NIA2–3 plants. The experiments were repeated three times with similar results. Each data point represents the mean ± SD (n = 3). Asterisks indicate a significant difference relative to Col (Student’s t-test, ***P < 0.001). (b) Eight-day-old wild-type, cngc6, cngc6/35S::NIA2–1, and cngc6/35S::NIA2–3 seedlings grown at 22 °C were exposed to 45 °C (HS) or maintained at 22 °C (Control) for 60 min. The NO levels in the plants were then examined by fluorescence microscopy using roots stained with DAF-FM DA. Bar = 100 μm. (c) The relative DAF-FM fluorescence densities in the roots. The data presented are the means ± SE of measurements taken from five independent experiments with at least ten roots for each treatment. *P < 0.05 versus Col (Student’s t-test). (d) Seedlings grown at 22 °C were exposed to 45 °C (HS) or maintained at 22 °C (Control) for 100 min, then returned to 22 °C and photographed 5 days later. The clusters are as follows: 1, wild type; 2, cngc6; 3, cngc6/35S::NIA2–1; and 4, cngc6/35S::NIA2–3. (e) Survival ratios of the seedlings after HS treatment. The data presented are the means ± SE of at least five independent experiments with 50 seedlings per experiment. *P < 0.05 versus Col (Student’s t-test)