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We previously reported that nitric oxide (NO) functions as a signal in thermotolerance. To illustrate its relationship with hydrogen peroxide (H 2 O 2 ) in the tolerance of Arabidopsis (Arabidopsis thaliana) to heat shock (HS), we investigated the effects of heat on Arabidopsis seedlings of the following types: the wild type; three NADPH oxidase-defective mutants that exhibit reduced endogenous H 2 O 2 levels (atrbohB, atrbohD, and atrbohB/D); and a mutant that is resistant to inhibition by fosmidomycin (noa1, for nitric oxide-associated protein1). After HS, the NO levels in atrbohB, atrbohD, and atrbohB/D seedlings were lower than that in wildtype seedlings. Treatment of the seedlings with sodium nitroprusside or S-nitroso-N-acetylpenicillamine partially rescued their heat sensitivity, suggesting that NO is involved in H 2 O 2 signaling as a downstream factor. This point was verified by phenotypic analyses and thermotolerance testing of transgenic seedlings that overexpressed Nitrate reductase2 and NOA1, respectively, in an atrbohB/D background. Electrophoretic mobility shift assays, western blotting, and real-time reverse transcription-polymerase chain reaction demonstrated that NO stimulated the DNA-binding activity of HS factors and the accumulation of HS proteins through H 2 O 2 . These data indicate that H 2 O 2 acts upstream of NO in thermotolerance, which requires increased HS factor DNA-binding activity and HS protein accumulation.

To characterize the role of nitric oxide (NO) in the tolerance of Arabidopsis (Arabidopsis thaliana) to heat shock (HS), we investigated the effects of heat on three types of Arabidopsis seedlings: wild type, noa1(rif1) (for nitric oxide associated1/resistant to inhibition by fosmidomycin1) and nia1nia2 (for nitrate reductase [NR]-defective double mutant), which both exhibit reduced endogenous NO levels, and a rescued line of noa1(rif1). After HS treatment, the survival ratios of the mutant seedlings were lower than those of wild type; however, they were partially restored in the rescued line. Treatment of the seedlings with sodium nitroprusside or S-nitroso-N-acetylpenicillamine revealed that internal NO affects heat sensitivity in a concentrationdependent manner. Calmodulin 3 (CaM3) is a key component of HS signaling in Arabidopsis. Real-time reverse transcriptionpolymerase chain reaction analysis after HS treatment revealed that the AtCaM3 mRNA level was regulated by the internal NO level. Sodium nitroprusside enhanced the survival of the wild-type and noa1(rif1) seedlings; however, no obvious effects were observed for cam3 single or cam3noa1(rif1) double mutant seedlings, suggesting that AtCaM3 is involved in NO signal transduction as a downstream factor. This point was verified by phenotypic analysis and thermotolerance testing using seedlings of three AtCaM3-overexpressing transgenic lines in an noa1(rif1) background. Electrophoretic mobility-shift and western-blot analyses demonstrated that after HS treatment, NO stimulated the DNA-binding activity of HS transcription factors and the accumulation of heat shock protein 18.2 (HSP18.2) through AtCaM3. These data indicate that NO functions in signaling and acts upstream of AtCaM3 in thermotolerance, which is dependent on increased HS transcription factor DNAbinding activity and HSP accumulation.

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