一氧化氮调控植物硒代谢机制研究

Accumulated evidence demonstrates that nitric oxide (NO) are signals to participate in multiple physiological processes, including systemic acquired resistance, the hypersensitive response, leaf senescence, programmed cell death, stomatal closure, root gravitropism, cell wall development, flowering repression, and development. Selenium (Se) is an essential micronutrient for many organisms, including animals and humans. It is also a beneficial nutrient for many plants. Soil Se concentration and bioavailability vary with parent material and environmental conditions, and the distribution of Se in soils is usually heterogeneous and site-specific. As plants are the main source of dietary Se, plant Se metabolism is therefore important for Se nutrition of humans and other animals. However, the concentration of Se in plant foods varies between areas, and too much Se can lead to toxicity. It is imporant in the current understanding of Se in higher plants,as well as advances in the genetic engineering of Se metabolism, particularly for biofortification and phytoremediation of Se-contaminated environments. In this work , speciation of selenium in Arabidopsis thaliana which has different endogenous NO level is performed via size-exclusion chromatography and ion-pairing reversed phase chromatography using inductively coupled plasma mass spectrometry (ICPMS) and nano-electrospray ion trap mass spectrometry (nanoESI-ITMS) for detection. The organ-specific accumulation, spatial distribution, and chemical speciation of Se are investigated in using inductively coupled plasma mass spectrometry, microfocused x-ray fluorescence elemental and chemical mapping (mXRF), Se K-edge x-ray absorption near-edge structure (XANES) spectroscopy, and liquid chromatography-mass spectrometry (LC-MS). By using chemiluminescence technique, fluorescent imaging of endogenous NO (DAF-FM DA), NO emission from Arabidopsis thaliana seedlings are visualized. Real-time quantitative PCR analysis are used to study the roles of gene in sulfate/selenium transport and assimilation. The biotin-switch technique are used to study S-nitrosylation in sulfate/selenium transport and assimilatory enzymes. Our data will further suggest that NO are important mediators in the process of Se transport and assimilation in higher plants. Theoretical matter as well as a practical one will be promoted in the genetic engineering of Se metabolism, particularly for biofortification and phytoremediation of Se-contaminated environments by regulating NO level.

NO作为信号分子广泛参与了对植物生长发育和生理生化过程调节；Se是人体必需的微量元素，也是植物的有益元素。由于其分布在地球上极不均匀，获得富硒植物并对高硒污染环境进行有效生物修复具有重要理论和实践意义。本研究项目以不同内源NO水平模式植物拟南芥为材料；在研究方法上，运用了muXRF、XANES和HPLC-ICP-MS分析实验材料中硒累积、空间分布和化学种类，荧光探针、化学发光法测定NO，RT-PCR分析Se吸收、转运、代谢相关基因表达水平变化，生物素标记(biotin switch)方法研究拟南芥硒代谢相关蛋白S-亚硝酰化修饰；在研究路线上，采用抑制剂实验、NO供体与清除剂实验、突变体实验等相结合的办法，综合研究分析NO对植物Se吸收、代谢的调节机制。预期结果可深化人们对NO在植物中的作用模式认识并为通过生物工程手段改善植物Se富集和生物修复能力提供理论依据。

微量元素硒可以作为植物体内的有益元素，对植物的生长发育具有重要影响。多数研究指出，较低浓度的硒可刺激植物生长，而高浓度却对植物造成毒害从而抑制其生长。一氧化氮作为一种重要的生物信号分子，在动植物体内起着至关重要的作用。已有部分研究表明硒能引起植物体内NO的变化，且NO能缓解硒诱导的水稻脂质过氧化，但目前尚缺乏关于NO对硒吸收代谢调控的分子机制研究。本研究以重要的粮食作物水稻为材料，以亚硒酸钠为硒源，以硝普钠作为NO供体，应用ICP/MS、HPLC-ICP/MS、qRT-PCR等研究手段，结合生长指标和生理生化指标测定，首次从基因水平上研究了一氧化氮调控水稻硒吸收和代谢的分子机理。. 研究显示，硒浓度<20 μM能促进水稻 “佳辐占”幼根生长，而≥20 μM却抑制根生长；根部硒累积水平与硒处理浓度之间显著正相关；硒浓度高于20 μM时，地上部分硒累积表现出饱和现象。在6 μM 硒处理下，10 μM SNP能有效促进水稻根部硒累积且不影响硒向地上部分的转运。外源NO清除剂cPTIO和NR抑制剂Tungstate处理均降低了根内NO含量和硒含量，但NO合成酶抑制剂L-NNA对NO含量及硒累积没有影响；暗示水稻根系中NO的合成主要是通过NR途径。HPLC-ICP/MS分析结果显示，NO提高了根部总水溶性含硒化合物含量，促进了SeCys及MeSeCys生物合成，而对SeMet的累积没有影响。. 我们进一步对硒吸收、代谢相关酶活性及基因表达水平进行了分析，得出了NO促进水稻根部Se吸收累积的可能机理：细胞内NO水平上升，促进了细胞膜上硒相关转运子OsSultr1;2、OsSultr 4;1及OsPT2表达上调，加强了根细胞对硒的吸收；基因Osγ-ECS、OsGS表达上调促进了GSH累积，后者可以促进无机硒转化为有机硒，从而进一步提高植物对无机硒的吸收速率；NO诱导的半胱氨酸合酶CS活性上升促进了硒代谢中间产物SeCys的累积；其甲基化产物MeSeCys的合成增强；另一方面，OsHMT表达上调，但SeMet含量并无明显增加，暗示SeMet可能通过参与含硒蛋白合成而转化。NO通过调控硒的吸收及代谢两个途径，促进了水稻根部对硒的累积。这些结果为研究植物硒吸收代谢和探索提高植物硒含量策略提供了理论依据。