Nitric Oxide Plays A Role In The Copper-induced Oxidative Damage In Highland Barley

As a very important signaling molecule,nitric oxide(NO)is involved in the various physiological responses in plants,including seed germination,primary and lateral root growth,flowering,pollen-tube growth regulation,fruit ripening,senescence,defense response and so on,especially in abiotic stresses.Copper(Cu)is a trace element that is essential for plants.However,in the excessive concentrations,this metal becomes powerfully toxic for plants.As a result of mining,smelting and other activities,Cu contamination is widespread.In this study,we investigate how NO works under copper stress in highland barley seedlings(Kunlun 12).The main results are as follows:1.The results show that excess copper inhibits the growth of highland barley seedlings in the different concentrations(100,200,400,800 μM),i.e.,a decrease in fresh weight,chlorophyll content,length of leaves and primary roots.Moreover,the underground part of highland barley seedlings is more sensitive to copper which can be significantly inhibited by 100 μM copper.The length of root reduces by 51.3%,while fresh weight reduces by 24.9%compared with control.2.Copper stress affects the root structure of highland barley,causing a reduction in the number of lateral roots.High concentration of copper(800 μM)almost completely blocks the formation of lateral roots.By detecting the content of copper in seedlings,it shows that copper in roots was significantly increased compared with control.The results of fluorescent probes prove that the production of NO burst in an early stage(12 h),while H2O2 rise significantly at 24 h with an accumulation of O2-.3.Copper induced the accumulation of intracellular antioxidants,such as reduced glutathione(GSH)and ascorbate(Asc).The detection of propidium iodide(PI)stain and Malondialdehyde(MDA)content indicate that copper induced lipid peroxidation.DAPI and TUNEL dyeing show that copper stress induce programmed cell death(PCD)in the root tips of highland barley.The accumulation of MDA content indicates a rise in lipid peroxidation,which demonstrates that the increased ROS lead to oxidative damage.4.By application of NR and NOS inhibitors,we find that the production of NO induced by copper stress is mainly from NR.Inhibition of NR activity can relieve the toxic effect of copper stress on highland barley,particularly for the underground part.The activity of NR is markedly induced by copper stress.The co-treatment of copper and SNP(NO donor)has combined effect on NR activity,while the addition of cPTIO(a scavenger of NO)and Tungstate(an inhibitor of NR)make the NR activity recover to control level.5.NBT staining shows that the changing of O2-is similar to H2O2.Copper stress and application of SNP induce the accumulation of O2-,while Tungstate and cPTIO can reduce the production of O2-significantly compared with copper stress.The exogenous application of SNP can cause a similar growth inhibition and H2O2 accumulation to copper stress,while NO scavenger treatment can obviously alleviated the toxicity effect induced by copper in highland barley seedlings.In summary,the production of NO stimulated by copper stress induces the accumulation of ROS,therefore,leads to the oxidative damage and inhibits the growth of highland barley seedlings.