Glutamine Synthetase Is a Molecular Target of Nitric Oxide in Root Nodules of Medicago truncatula and Is Regulated by Tyrosine Nitration
Glutamine Synthetase Is a Molecular Target of Nitric Oxide in Root Nodules of Medicago truncatula and Is Regulated by Tyrosine Nitration
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DOI：
10.1104/pp.111.186056
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发表时间：
2011-11-01
期刊：
影响因子：
7.4
通讯作者：
Carvalho, Helena G.
中科院分区：
文献类型：
--
作者：
Melo, Paula M.;Silva, Liliana S.;Carvalho, Helena G.
一氧化氮（NO）是根瘤菌-豆科植物共生过程中的重要调控因子，但其在根瘤功能中的生物学作用尚不清楚。为了解开信号转导级联和最终NO功能，有必要确定其分子靶点。研究结果表明，根瘤代谢的关键酶谷氨酰胺合成酶（GS）是蒺藜苜蓿根瘤中NO的分子靶点，受酪氨酸（Tyr）硝化的调控，与根瘤的主动固氮作用有关。在体外研究中，使用纯化的重组酶产生的大肠杆菌，证明了M。蒺藜根瘤GS同工酶（MtGS 1a）通过Tyr硝化作用受到NO介导的失活，并确定Tyr-167为酶失活的关键调节硝化位点。使用夹心酶联免疫吸附试验，它表明，GS是硝化在植物和硝化状态的变化有关的主动固氮。在无效的结节和结节喂硝酸盐，两个条件下，其中固氮受损和GS活性降低，一个显着增加结节GS硝化水平进行了观察。此外，处理根瘤与NO供体硝普钠导致增加体内GS硝化伴随着GS活性的降低。我们的研究结果支持一氧化氮在调节根瘤氮代谢中的作用，并将GS作为一个重要的球员在这个过程中。我们认为，NO介导的GS翻译后失活与代谢产物通道有关，以增强根瘤对NO的抗氧化防御。
Nitric oxide (NO) is emerging as an important regulatory player in the Rhizobium-legume symbiosis, but its biological role in nodule functioning is still far from being understood. To unravel the signal transduction cascade and ultimately NO function, it is necessary to identify its molecular targets. This study provides evidence that glutamine synthetase (GS), a key enzyme for root nodule metabolism, is a molecular target of NO in root nodules of Medicago truncatula, being regulated by tyrosine (Tyr) nitration in relation to active nitrogen fixation. In vitro studies, using purified recombinant enzymes produced in Escherichia coli, demonstrated that the M. truncatula nodule GS isoenzyme (MtGS1a) is subjected to NO-mediated inactivation through Tyr nitration and identified Tyr-167 as the regulatory nitration site crucial for enzyme inactivation. Using a sandwich enzyme-linked immunosorbent assay, it is shown that GS is nitrated in planta and that its nitration status changes in relation to active nitrogen fixation. In ineffective nodules and in nodules fed with nitrate, two conditions in which nitrogen fixation is impaired and GS activity is reduced, a significant increase in nodule GS nitration levels was observed. Furthermore, treatment of root nodules with the NO donor sodium nitroprusside resulted in increased in vivo GS nitration accompanied by a reduction in GS activity. Our results support a role of NO in the regulation of nitrogen metabolism in root nodules and places GS as an important player in the process. We propose that the NO-mediated GS posttranslational inactivation is related to metabolite channeling to boost the nodule antioxidant defenses in response to NO.