一氧化氮调控棉花适应淹水胁迫的机制

Waterlogging is a major hazardous abiotic stress which has imposed a great challenge to cotton growth and yield. It is of great importance to alleviate the damages in cotton plants subjected to waterlogging stress. NO has been demonstrated to be involved in plant adaptive responses to environmental constraints, but the mechanism of NO regulating cotton adaptation to waterlogging stress remains unknown. In this study, cotton (c.v K836 and Lumian532) will be grown under a rain-shelter or in potted soil in a greenhouse, and subjected to 5-10 day-waterlogging at flowering stage. The 15N isotopic tracer of NO donor sodium nitroprusside (SNP) and the NO scavenger 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethyl-imidazoline-1-1-oxyl- 3-oxide (Carboxy-PTIO) were applied to leaves or roots of cotton to examine the effects of NO on waterlogging adaptation and the underlying mechanism. The physiological events will be investigated by examining plant biomass and partitioning, photosynthesis, activity of anaerobic enzymes and levels of endogenous hormones in cotton plants. More importantly, the absorption, transport, distribution and metabolism of NO will be studied by radionuclide tracing techniques. On this basis, gene expression in the roots and leaves of each treatment will be analyzed through solexa sequencing method. Genes related to synthesis, metabolism and signal transduction of NO and all gene expression differences in roots and leaves under different treatments will be determined by analyzing cotton genome data using bioinformatics methods. Thus the mechanism of NO regulating cotton adaptation to waterlogging stress will be further documented at the molecular level.

淹水胁迫影响棉花生长发育，持续淹水引起棉花减产甚至绝产，缓解淹水胁迫对棉花抗灾夺丰收至关重要。一氧化氮（NO）是重要的气体信号分子，它在植物抗逆响应中发挥着关键作用，但其缓解棉花涝渍伤害的机制尚不清楚。本项目拟以2个棉花品种（K836和鲁棉532）为材料，在田间电动遮雨棚下旱池内种植，同时温室内盆栽，于花铃期给予5-10d的淹水处理，期间叶面喷施或根施15N标记的一氧化氮外源供体硝普钠（SNP）与一氧化氮清除剂cPTIO，以未喷施的棉花为对照，研究NO对棉花淹水伤害的缓解效应，以及NO对受淹棉花厌氧代谢酶活性、相关激素含量等的影响；重点通过放射性同位素15N示踪技术，研究NO在受淹棉花体内的吸收、转运、分布和代谢，明确其作用路径。在此基础上，通过表达谱和qRT-PCR分析，研究NO合成、代谢及信号转导关键基因的差异表达规律，深入揭示NO作为信号分子调控棉花适应淹水胁迫的生理和分子机制。

本项目以棉花（Gossypium hirsutum L.）品种K836为材料，在山东棉花研究中心试验站（临清，115°42'E, 36°61'N）的电动遮雨棚内，通过喷施一氧化氮（Nitric oxide, NO）调节剂（NO供体SNP或NO清除剂cPTIO）处理花期淹水棉株，深入研究了NO在棉花淹水胁迫响应中的作用及其机理。试验设置不淹水对照（NWL，正常灌溉以维持土壤含水量为最大持水量的60~70%）、淹水对照（WL，淹水10 d）、淹水并喷施SNP（WLS，淹水10 d，淹水期间喷施500 µmol/L SNP）和淹水并喷施cPTIO（WLP，淹水10 d，淹水期间喷施500 µmol/L cPTIO）4个处理，研究了NO对受淹棉花生长发育、经济产量、产量结构和纤维品质等的影响，旨在探明NO对淹水棉花生长发育和产量的影响；测定了NO调节剂处理后，受淹棉花叶片光合作用、抗氧化酶系统活性、厌氧代谢酶活性、碳氮代谢和内源激素含量等的变化，旨在揭示NO在棉花淹水胁迫响应中的作用机制；通过实时定量PCR 检测乙醇脱氢酶（GhADH）等无氧呼吸相关基因，乙烯合成（GhACS8）和乙烯响应的转录因子基因（ERF）、ABA 合成基因（GhNCED2）和赤霉素合成相关基因（GhGA3ox2）等激素合成相关基因和一氧化氮相关基因（GhNIR）的差异表达情况，旨在揭示NO在棉花淹水胁迫响应中的作用机理。我们发现，淹水棉株喷施NO调节剂有效改变了棉株NO含量，其中喷施SNP显著提升了棉株NO含量，在一定程度上抑制了厌氧代谢酶活性，缓解了细胞膜脂过氧化程度，调节了受淹棉株体内相关基因表达，协调相关激素代谢平衡，使受淹棉株光合能力得到恢复。与之相反，喷施cPTIO则显著降低了棉株NO含量，加重了膜系统和光合系统的破坏，生长发育得到进一步抑制，减产幅度增大。因此，提高受淹棉株NO含量，可有效缓解受淹棉株细胞膜脂过氧化和厌氧呼吸，诱导激素合成代谢相关基因表达，维护激素平衡，促进光合生产能力恢复，一定程度上提高了棉花对淹水胁迫适应能力，减少产量损失。因此，NO在分子、生理和植株水平上对棉花响应淹水起着重要的调控作用。