No Ctk Delay Effects And Mechanisms Of Wheat Leaf Senescence Process

Nitirc oxide (NO) is a ubiquitous messenger and effective molecule in biology. It plays an important role in both animals and plants. It is described as a multifunctional second messenger in animals since it plays various functions including the relaxation of blood vessels, neurotransmission, gene experession and defense during immune response etc. The research of NO in plant is later than animals. But lots of evidence suggest that NO participates in a series of key physiological processes such as leaf and fruit senescence, leaf and root development, seed germination, stomatal movement, programmed cell death and responses to biotic and abiotic stesses. Cytokinins (CTK) are important plant growth substances which play an important role in delaying the leaf senescence. Although the research has indicated that NO and CTK all can inhibite the leaf senescence, information is still lacking regarding whether the NO signaling is involved in the cytokinin-inhibited leaf senescence. In the present study, to reveal the role and it's mechanism of NO in CTK-inhibited leaf senescence, we used wheat (Triticum aestivum L.) seedlings as plant material, treated the isolated wheat leaves with NO scavenger hemoglobin (Hb), NR inhibitor sodium tungstate (Na₂WO₄) or NOS inhibitor L-NAME combined with kinetin (KT) or zeatin (ZT) treatment, and various physiological and biochemical parameters were measured, the main results were presented as follows:1. Both KT and ZT significantly inhibited the decreases in contents of photosynthetic pigment and soluble protein, and the increase in malondialdehyde content, indicating isolated wheat leaf senescence can be delayed not only by plant native ZT, but also by artificial KT; However, when wheat leaves were treated with KT or ZT in the presence of NO scavenger Hb, NR inhibitor Na₂WO₄ or NOS inhibitor L-NAME, the above effects of KT or ZT were obviously reduced. It is suggested that CTK-inhibited senescence of isolated wheat leaf is mediated by NO which is probably produced by NR and NOS-like enzyme.2. Compared to control treatment, both KT and ZT significantly enhanced activities of NR and NOS. However, when wheat leaves were treated with KT or ZT in the presence of Na₂WO₄ or L-NAME, the KT- and ZT-induced increases in activities of NR and NOS were respectively inhibited by Na₂WO₄ and L-NAME. The results show that CTK can induce the enhancement of NR and NOS activities during CTK-inhibited senescence of isolated wheat leaf, and that the used concentration of inhibitor Na₂WO₄ or L-NAME can significantly inhibit the activity of NR and NOS-like enzyme respectively.3. By using the NO-specific fluorescent probe DAF-2DA and laser-scanning confocal microscopy, the endogenous NO level in isolated wheat leaves was directly measured and the results showed that the NO level in wheat leaves under KT or ZT treatment was obviously higher that that under control. However, when wheat leaves were treated with KT or ZT in the presence of Hb, Na₂WO₄ or L-NAME. the KT- and ZT-induced increases of NO level were respectively inhibited by Hb, Na₂WO₄ and L-NAME.These results directly indicated that NR- and NOS-dependent NO production was indeed induced by KT and ZT in wheat leaves.The above results combined together indicate that NO as signaling molecular is involved in the signal transduction pathway of CTK-inhibited senescence of isolated wheat leaves and that CTK-induced NO generation is dependent on NR and NOS-like enzymes.4. The activities of SOD, CAT, POD and APX in wheat leaves under KT or ZT treatment were observably higher than that under the control, while the H₂O₂ content under KT or ZT treatment was obviously lower than that under the control, indicating that CTK inhibits senescence of wheat leaves by enhancing the activities of antioxidant enzymes and thereby increasing the scavenging capacity of reactive oxygen species (ROS) in wheat leaves.However, when wheat leaves were treated with KT or ZT in the presence of Hb, Na₂WO₄ or L-NAME, the activities of SOD, CAT, POD and APX in wheat leaves were significantly lower and H₂O₂ content was obviously higher than that in KTor ZT treatment, suggesting that NO mediates the CTK-inhibited senescence of wheat leaves by increasing the activities of antioxidant enzymes to scavenge the ROS in wheat leaves.