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Due to the global warming, rising temperature became a serious threat to the world agriculture. Faba bean plant is very susceptible to heat stress and performs poorly under high temperature condition. Therefore, the performance of faba bean (Vicia faba L.), genotype 'C5' was tested to explore (i) the biosynthesis of proline (Pro) in response to heat stress (HS) and the involvement of nitric oxide (NO) and Pro in thermotolerance mechanism and (ii) the role of heat shock proteins (HSPs) and Pro in defense, and their correlation with the tolerance of faba bean plant to HS. Exposure of plants to HS for 48 h resulted in increased levels of reactive oxygen species [hydrogen peroxide (H2O2) and superoxide ions (O-2(center dot-))), nitric oxide (NO), accumulation of Pro and malondialdehyde, electrolyte leakage, DNA damage, chlorophyll (Chi) degradation and in decreased leaf relative water content (RWC) and Chl content in faba bean plants. However, at the same time increasing levels of HS (32, 37 and 42 degrees C) increased the accumulation of NO, Pro, activities of Pro metabolizing enzymes (Delta(1)-pyrroline-5-carboxylate synthetase, ornithine-delta-aminotransferase and proline dehydrogenase) and expression of HSPs (Hsp17.6, Hsp70, Hsp90-1, and Hsp101). Induced biosynthesis of NO and Pro under HS activated defense system of plants by enhancing the activities of antioxidant enzymes [superoxide dismutase, catalase, glutathione reductase and ascorbate peroxidase] and also by increasing the content of reduced glutathione (GSH), ascorbate (AsA) and oxidized glutathione (GSSG). While, an increase in GSSG content was found lesser than an increase in GSH under HS stress. Application of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO; an NO scavenger) decreased the accumulation of leaf RWC and Pro and NO. Therefore, it can be proposed that NO acts as both signal molecule and promotor of Pro biosynthesis. The present investigation reveals that above studied parameters could be used in identifying a HS-tolerant plant that can be utilized in future breeding programs.