Arginine Metabolism And The Roles Of NO In Root Development And Stress Response In Malus Hupehensis (Pamp) Rehd. Var Pinyiensis Jiang

The coordinate formation of nitric oxide (NO) and polyamines through arginine metabolism, as well as NO biosynthetic pathways and the physiological roles of NO in root development and abiotic stress response were investigated in Malus hupehensis (Pamp) Rehd. var pinyiensis Jiang, using water-cultured seedlings and potted trees. The results are as follows.1.A luminol-H2O2 chemiluminescence method was developed to determine NO contents and nitric oxide synthase (NOS) activities in plants. Experiments with exogenous L-arginine, NOS inhibitors, NO scavenger MB and inactivation of enzymes by boiling samples proved that it is an effective method for assaying NO contents and NOS activities in plants.2.Higher levels of arginine are located in roots than in stems and leaves with lignified roots being the highest in young Malus hupehensis (Pamp) Rehd. var pinyiensis Jiang trees. Arginine content in lignified roots reaches the highest during leaf expansion, and then gradually decreases with the development of shoots and leaves. NO contents and enzymatic sources for NO formation differ greatly in different organs of young Malus hupehensis (Pamp) Rehd. var pinyiensis Jiang trees. Roots have the highest levels of NO content, NOS and NI-NOR activities, and tender stems follows, with leaves being the lowest. Root and tender stem mitochondria but not leaf mitochondria can reduce nitrite to NO in the presence of NADH as an electron donor. In arginine metabolism, polyamine pathway, NO pathway and the content of arginine are positively correlated in a tissue-specific manner. Higher levels of arginine, putrescine, spermidine, spermine and NO, as well as arginase, ADC, ODC and NOS activities exist in fine roots than in leaves, with white roots and juvenile leaves being higher than brown roots and mature leaves, respectively. The results shows that arginine metabolism in roots is more active than in leaves.3.The levels of arginine and arginase in cotyledons are higher than in roots and stems, and decreased slowly with the seedling development in Malus hupehensis (Pamp) Rehd. var pinyiensis Jiang. From 8 to 20 days of growth, higher levels of NO and NOS exist in stems than in roots and cotyledons, and reach the highest after 11 days of growth, coinciding with the rapid elongation of the stems, while those of roots are the lowest and reaches the highest after 8 days growth, coinciding with the development of lateral roots. NO and NOS levels in leaves reach the highest after 17 days growth, coinciding with the rapid expansion of leaves. The highest levels of arginine, NO and the two enzymes were detected in cotyledons after 5 days growth, indicating that arginine metabolism play important roles in the early development the seedling.4.In arginine metabolism, exogenous L-arginine (3 mmol/L) improves NO formation significantly in roots of Malus hupehensis (Pamp) Rehd. var pinyiensis Jiang seedlings under 100 mmol/L NaCl stresses, while 3 mmol/L urea decreases NO formation. Application of 1mmol/L spermine decreases NO formation and NOS activities in roots of Malus hupehensis (Pamp) Rehd. var pinyiensis Jiang seedlings with or without 100mmol/L NaCl stress. Application of 0.1mmol/L nitrate reductase inhibitor NaN3, 50mmol/L NOS inhibitor AG or 5mmol/L ADC inhibitor D-arginine for 12h inhibits NO formation greatly in detached white roots of Malus hupehensis (Pamp) Rehd. var pinyiensis Jiang, while 50mmol/L arginase inhibitor KF improves NO formation greatly.5.Exogenous NO improves root formation in a dose-dependent manner in Malus hupehensis (Pamp) Rehd. var pinyiensis Jiang seedlings. SNP concentrations of 10μmol/L and 50μmol/L are most effective in increasing root length and root number, respectively. 100μmol/L IBA induces a transient increase in endogenous NO levels at early (15~30 min) and late (48~96 h) stages of IBA treatment in roots of Malus hupehensis (Pamp) Rehd. var pinyiensis Jiang seedlings and large amounts of LRP appear at 96h after IBA treatment. The IBA-induced NO increase is inhibited by 100μmol/L Quercetin (a protein kinase inhibitor), suggesting that NO acts as an important messenger in IBA-induced rooting process and protein kinase is upstream of its generation.6.Thirty percent of PEG-6000 induces a transient increase in NO content in roots at 0.5h and 1.5h after treatment, which is associated with the rapid increase in cytosolic NOS activities and nitrite-reducing activities at the expense of NADH in root mitochondria. In contrast to the NO levels, ROS levels are always higher in roots of PEG-treated seedlings, especially at the late stages of osmotic stress.7.Exogenous NO pretreatment at different concentrations of 10, 100, and 1000μmol/L for 48h improves salt resistance in Malus hupehensis (Pamp) Rehd. var pinyiensis Jiang seedlings under 0.35% NaCl stress, characterized by improved fresh weight increment. SNP concentration of 100μmol/L is the most effective, follows 1000μmol/L, and 10μmol/L is the least. Exogenous NO pretreatment for 48h might have reduced the'attack effect'caused by NaCl stress, characterized by decreased NOS activities in roots and increased NOS activities in leaves during pretreatment, and decreased NO content and NOS activities at 12h after NaCl stress in contrast to the control.8.Exogenous NO (100μmol/L SNP) can reduce ROS levels and malondialdehyde content, and improve SOD,CAT activities and proline content in detached leaves of Malus hupehensis (Pamp) Rehd. var pinyiensis Jiang during leaf senescence, but have no obvious effects on POD and APX activities. 100μmol/L SNP delays 30%PEG-6000 induced leaf senescence with improved chlorophyll, proline, ROS, CAT, POD and APX levels, and decreased malondialdehyde content, but have no effects on SOD activity.