Asparagine metabolism and function in nitrogen signaling

天冬酰胺代谢和氮信号传导中的功能

• 批准号: RGPIN-2018-05478
• 负责人: Marsolais, Frédéric
• 金额: $ 3.42万
• 依托单位: University of Western Ontario
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=713700
• 关键词: Asparagine; metabolism; function; nitrogen; signaling

The amino acid asparagine is a major form of nitrogen commonly transported to sink tissues of higher plants, including roots, flowers and developing seeds, due to its high nitrogen to carbon ratio. Local biosynthesis of asparagine in reproductive tissues can also pre-condition storage reserve accumulation in grain. In leaf, asparagine is accumulated transiently at night when, in the absence of photosynthesis, levels of organic acids are insufficient for nitrogen assimilation. Asparagine is an important intermediate in photorespiration, providing an input of nitrogen to balance an output of serine or glycine used in other pathways. The objectives of this proposal are to achieve a detailed understanding of asparagine metabolism and of the function and perception of asparagine as a metabolite signal of organic nitrogen in higher plants. Reporter screens with previously identified asparagine-responsive genes will help identify factors involved in their regulation. Gene regulatory networks will also be investigated by in silico promoter analysis, identification of cognate regulators and inducible gene profiling assays. The biochemistry of the photorespiratory pathway of asparagine metabolism will be investigated. The enzyme omega-amidase is involved in the breakdown of the reaction product of asparagine transamination, alpha-ketosuccinamate and of its reduced form, alpha-hydroxysuccinamate. It shares a high degree of sequence identity with an enzyme catalyzing the breakdown of a damaged form of the antioxidant glutathione, arising from transamination. The structural determinants of substrate specificity in these enzymes will be identified by an approach combining protein crystallography and site-directed mutagenesis. The dehydrogenase enzyme responsible for the reduction of alpha-ketosuccinamate will be identified. Two alternative approaches will be used. First, a bioinformatic approach will examine co-expression of asparagine metabolic and photorespiratory genes with dehydrogenases in the genome of the model plant Arabidopsis thaliana. Candidate genes will be characterized by determining the catalytic activity of the corresponding recombinant enzyme in vitro. The second approach will involve purification of the enzyme from soybean leaves, and identification of the purified protein by digestion with a site-specific protease, mass spectrometry and search against a proteome database. Candidate genes will be validated by profiling metabolites in the corresponding knock-out mutant of A. thaliana in response to treatment with exogenous non-labeled and stably labeled asparagine. The research results will improve our understanding of asparagine metabolism and signaling in higher plants.

氨基酸天冬酰胺是氮的主要形式，通常运输到高等植物的库组织，包括根，花和发育中的种子，由于其高的氮碳比。生殖组织中天冬酰胺的局部生物合成也是籽粒贮藏物质积累的先决条件。在叶片中，天冬酰胺在夜间短暂积累，在没有光合作用的情况下，有机酸水平不足以进行氮同化。天冬酰胺是光呼吸中的重要中间体，提供氮的输入以平衡用于其他途径的丝氨酸或甘氨酸的输出。本提案的目的是实现天冬酰胺代谢的功能和感知的天冬酰胺作为一种代谢信号的有机氮在高等植物中的详细了解。与以前确定的天冬酰胺反应基因的报告屏幕将有助于确定参与其调节的因素。基因调控网络也将通过计算机启动子分析、同源调控因子的鉴定和诱导型基因分析来研究。天冬酰胺代谢的光呼吸途径的生物化学将被研究。酶ω-酰胺酶参与天冬酰胺转氨作用的反应产物α-酮基琥珀酰胺酸及其还原形式α-羟基琥珀酰胺酸的分解。它与催化转氨作用产生的抗氧化剂谷胱甘肽受损形式分解的酶具有高度的序列同一性。在这些酶的底物特异性的结构决定因素将确定结合蛋白质晶体学和定点诱变的方法。将鉴别负责α-酮基琥珀酸还原的脱氢酶。将采用两种备选办法。首先，生物信息学方法将检查模式植物拟南芥基因组中天冬酰胺代谢和光呼吸基因与天冬酰胺酶的共表达。候选基因将通过测定相应重组酶的体外催化活性来表征。第二种方法将涉及从大豆叶中纯化酶，并通过用位点特异性蛋白酶消化、质谱分析和对蛋白质组数据库的搜索来鉴定纯化的蛋白质。候选基因将通过分析相应的A基因敲除突变体中的代谢产物来验证。拟南芥对外源非标记和稳定标记天冬酰胺处理的响应。研究结果将有助于我们更好地了解高等植物中天冬酰胺的代谢和信号转导。