18Heavy metal stress limits crop production through its effects on seed germination and 19 photosynthesis. Nitric oxide (NO), a versatile signaling molecule, plays a significant role in 20 heavy metal stress tolerance. In the present investigation, the efficacy of NO application in the 21 alleviation of copper (Cu) induced adverse impact on seed germination and photosynthesis of 22 mustard plant (Brassica juncea L.) was evaluated. Pretreatment with NO donor sodium 23 nitroprusside (SNP), significantly improved seed germination and alleviated Cu-accrued 24 oxidative stress in B. juncea seeds. However, in the absence of NO, Cu showed a higher 25 reduction in seed germination rate. Further, NO modulated the activities of antioxidant enzymes 26 and sustained the lower level of lipid peroxidation by reducing H 2 O 2, and thiobarbituric acid 27 reactive substances (TBARS), thereby elevated the antioxidative capacity in Cu-exposed seeds.
28Seeds pretreated with NO also retained higher amylase activities for the proper seed germination 29 when compared with control. NO mitigated Cu toxicity through an improved antioxidant system, 30 and reducing Cu-induced accumulation of reactive oxygen species (ROS), reduction in lipid 31 peroxidation improving photosynthetic efficiency and growth of the mustard plant. It may 32 concluded that NO improved amylase activity, modulated activity of antioxidant enzymes, and 33 enhanced the germination rate seeds under Cu stress, thereby improved photosynthesis and 34 growth.35 Introduction 36Heavy metal pollution has been well-known since long time; however, its continuous use 37 becomes hazardous in respect of agricultural and human health, mostly in lesser developed 3 38 countries [1,2]. In this perspective, Cu became apparent as a severe pollutant because of its 39 extensive use in industries and as a pesticide in agriculture [3,4]. The metal Cu performs a 40 remarkable array of functions in living organisms that are essential for life. Proteins that contain 41 Cu as cofactor perform various biochemical processes which are involved in plant growth and 42 development, and protective mechanisms [5]. For plant life, Cu is widely recognized as an 43 essential microelement [4]. Usually, the optimum levels of Cu in leaves are 10 µg g -1 dry mass 44 [6] and the acute level of toxicity found for most of the crop plants are slightly higher than 20-30 45 µg g -1 dry mass [7]. Phytotoxicity of Cu in leaves affects photosynthesis adversely by targeting 46 thylakoids; particularly PSII has been identified to be the main target [8]. Excessive 47 accumulation of Cu dissipates the electron transport in PS I and PS II, inhibits 48 photophosphorylation and alters membrane integrity [9,10]. Also, high concentrations of Cu 49 have been extensively reported to induce oxidative stress resulting in disruption of structure of 50 biological molecules and membranes by increasing lipid peroxidation [11,12]. Moreover, excess 51 Cu alters the proper functioning of mineral uptake[13,14]. Assay of seed germination is ...