加密信号（CryptoSignals）：揭示复杂蛋白质中隐秘的兼职位点的信号传导作用

该研究采用跨学科的方法来发现植物环核苷酸和一氧化氮（NO）通路中的新信号分子。拟定的研究目标是发现和表征1）植物核苷酸环化酶（鸟苷酸环化酶、GC、腺苷酸环化酶、AC）；2）磷酸二酯酶（PDE）以及3）NO传感器血红素蛋白。鉴于植物蛋白已经进化出复杂的多域组织，采用常规基于同源性的方法无法识别这些信号传导分子。在本课题中，首先，通过构建氨基酸序列搜索基元，结合计算和结构方法，发现新型的GC、AC、PDE和NO传感蛋白。其次，用包括酶和免疫分析、基因诱变、LC-MS、比较基因组学和系统分析等实验方法，表征它们的活性，并揭示它们如何在植物细胞的局部微环境中实现一个信号通路到另一个信号通路的动态和快速时空切换。第三，将在拟南芥和其他模式植物或农作物植物中进行生理学和表现型研究，以确定这类新的信号分子是否可以作为生物技术革新富有吸引力的靶标，从而有助于生产胁迫耐受粮食作物。

To enable plant biotechnological innovations, a comprehensive understanding of the signaling pathways in the plant cell is required, but the generating (guanylate cyclases, GCs; adenylyl cyclases, ACs) and degrading (phosphodiesterases, PDEs) enzymes of cyclic mononucleotides (cGMP/cAMP), and the heme-based protein sensors of nitric oxide (NO), remained largely elusive. cGMP/cAMP and NO are key signaling molecules that regulate diverse biological processes in organisms across the tree of life. Their biological roles include mediating plant growth and developmental processes, as well as responses to biotic and abiotic stresses including light, heavy metals, temperature, salinity, drought, and ozone. Regular homology-based approaches failed to identify the cellular components of these signaling molecules because plant proteins have evolved complex multi-domain organizations that mask these seemingly cryptic functional sites which is made more complicated still if they have diverged beyond the detection limits of BLAST and antibodies raised in animals or bacteria. Rather than a stand-alone molecule, many plant proteins have complex domain organizations consisting for instance of an extracellular ligand recognition receptor region, a single or multi-pass transmembrane region and a cytosolic region that may accommodate one or indeed several functional domains with protein-protein interaction or catalytic roles. These signaling molecules are poorly understood and even controversial in the field of plant cell signaling because they have seemingly low activities compared to the corresponding proteins in animals and bacteria, hence cryptic, but modern high resolution detection methods have established beyond doubt their functionalities both in vitro and in planta. These “CryptoSignals” however have profound effects on the molecular and physiological functions. Thus, how moonlighting sites of such nature exert regulatory roles within and between proteins to mediate a myriad of biological processes and responses have attracted increasing attention. In this research, amino acid search motifs will be built or refined and various modern computational and structural approaches including bioinformatic tools, webservers, and databases as well as structural modeling and simulations, will be developed or employed, to enable the identification of novel GCs, ACs or PDEs, and NO sensing proteins in plants. Unlike homology-based approaches, only amino acids that have direct functional roles at the catalytic centers or heme-binding region of the corresponding proteins in organisms across species, will be included in the search motifs. A combination of multi-disciplinary yet complementary approaches such as enzyme- and immuno-assays, mutagenesis, LC-MS/MS, comparative genomics, and systems analysis, will be employed to characterize their functions. This study will reveal how moonlighting sites of this nature regulate other primary domains to enable dynamic and rapid spatiotemporal switching of one signaling pathway to another in the localized micro-environment of the plant cell. To elucidate their biological roles, detailed physiology and phenotyping studies in Arabidopsis thaliana and other model or crop plants, will be conducted. A highly localized approach that concentrates on bioengineering of regulatory genes for signaling cascades such as those in the cyclic mononucleotide and NO signaling pathways and, in a stimulus-specific manner, can overcome problems of metabolic imbalance commonly associated with conventional overexpression of genes, which is unprofitable and has in the past, hampered the application of transgenic crop plants in fields. The identified candidates will constitute a new class of signaling molecules with unique architecture and domain combinations and can act as attractive targets for genetic manipulations to confer increased tolerance to abiotic and biotic stresses.