甲烷诱导黄瓜不定根发生的分子机理研究

Our recent results showed that methane (CH4) might be a new gaseous signal molecule. For example, auxin was able to stimulate CH4 releasing in cucumber explants. The application of methane-rich water (MRW) not only mimicked the releasing of endogenous CH4, but also triggered cucumber adventitious rooting. The involvement of auxin signaling related genes and cell cycle regulatory genes was also suggested. Further results revealed that nitric oxide (NO) might be the downstream molecular in this event. To explore this possibility, in this project, the possbility of reactive oxygen species (ROS)- and methionine (Met)-mediated auxin-triggered CH4 production and corresponding relationship with adventitious rooting were investigated in cucumber explants. The enzymatic- and non-enzymatic resources of NO synthesis in CH4-mediated process were also analyzed. By using electron spin resonance (ESR) and laser scanning confocal microscope (LSCM), combined with the application of the NO donors, scavenger and possible synthetic inhibitors, the spatial/temporal of NO distribution/content, the transcripts of cell cycle regulatory genes, as well as the changes of adventitious root primordia and adventitious root formation, were compared. The classification of S-nitrosylated proteins and the identification of the S-nitrosylated cysteine residues were also carred out with site-specific S-nitrosoproteomic approach. Therefore, the downstream components and signal transduction pathway (especially related to NO), and its relationship with auxin signaling were summarized. These results would not only provide the knowledge of biological roles of CH4 in plants, but also provide theoretical basis for the development of new crop growth regulators.

我们最近发现，甲烷（CH4）可能是一种新的气体信号分子；生长素能诱导黄瓜外植体下胚轴基部CH4的产生，模拟内源CH4释放的富甲烷水通过调节生长素应答基因以及细胞循环周期基因表达来诱导不定根发生，一氧化氮（NO）可能是其下游的信号分子。为了探查相关作用机制，本课题首先从活性氧和甲硫氨酸两个方面探查生长素诱导的CH4生物合成途径及其与不定根发生的关系，分析受CH4调控的黄瓜不定根发生过程中NO来源以及时空变化，结合采用清除剂以及抑制剂，比较细胞循环周期基因以及不定根原基/不定根发生的差异，明确CH4诱导的NO与不定根发生的关系；采用位点专一性的亚硝基蛋白质组学方法鉴定NO介导的S-亚硝基化蛋白种类和氨基酸靶点，从而以NO信号转导为重点探查CH4调控不定根发生的下游关键蛋白、信号通路以及与生长素信号途径的关系。上述结果不仅提供植物CH4生物学效应的证据，同时也为开发新的生长调节剂提供初步的知识。

我们先前的研究发现甲烷（CH4）可以诱导黄瓜不定根的产生，但是并未完全阐明下游的靶分子。已经知道，一氧化氮（NO）作为一种常见的气体信号分子可以参与根形态建成，因此本课题首先通过探究了NO作为CH4下游信号分子参与黄瓜不定根发生的可能性。实验首先发现CH4和NO释放化合物SNP、NONOate均可诱导不定根的发生与生长，CH4可以诱导NO的产生，而NO清除剂PTIO和cPTIO则可以逆转此现象，同样的NO合成相关途径关键酶抑制剂Tungstate（硝酸还原酶抑制剂）和L-NAME（哺乳动物类一氧化氮合酶抑制剂）以及β-HEH（二胺氧化酶抑制剂）也能明显降低内源NO含量，逆转CH4对黄瓜不定根发育的诱导作用；进一步的研究发现此过程与负责不定根发育的标记基因，包括CsDNAJ-1、CsCDPK1、CsCDPK5细胞分裂相关基因CsCDC6，以及两个生长素信号基因CsAux22D-like和CsAux22B-like有关；进一步通过免疫印迹分析总s-亚硝基化蛋白，初步证明s-亚硝基化也参与CH4诱导黄瓜不定根的发育。此外，我们的进一步研究发现硫化氢（H2S）和谷胱苷肽（GSH）均可以作为下游信号分子参与甲烷诱导的不定根发生；过氧化氢（H2O2）可以诱导CH4的产生，从而参与不定根发生。最后，我们还获得了用于大田农业生产的一种富甲烷灌溉装置的实用新型专利。上述研究结果不仅部分阐明了CH4诱导黄瓜不定根发育的下游靶信号，也为不定根在营养吸收以及提高植株耐受性方面提供了理论和实践基础。