Masclaux-Daubresse Céline, Reisdorf-Cren Michèle, Pageau Karine, Lelandais Maud, Grandjean Olivier, Kronenberger Joceline, Valadier Marie-Hélène, Feraud Magali, Jouglet Tiphaine, Suzuki Akira
Unité de Nutrition Azotée des Plantes, Institut National de la Recherche Agronomique, 78026 Versailles cedex, France.
Plant Physiol. 2006 Feb;140(2):444-56. doi: 10.1104/pp.105.071910. Epub 2006 Jan 11.
Glutamate (Glu) metabolism and amino acid translocation were investigated in the young and old leaves of tobacco (Nicotiana tabacum L. cv Xanthi) using [15N]ammonium and [2-15N]Glu tracers. Regardless of leaf age, [15N]ammonium assimilation occurred via glutamine synthetase (GS; EC 6.1.1.3) and Glu synthase (ferredoxin [Fd]-GOGAT; EC 1.4.7.1; NADH-GOGAT; EC 1.4.1.14), both in the light and darkness, and it did not depend on Glu dehydrogenase (GDH; EC 1.4.1.2). The [15N]ammonium and ammonium accumulation patterns support the role of GDH in the deamination of [2-15N]Glu to provide 2-oxoglutarate and [15N]ammonium. In the dark, excess [15N]ammonium was incorporated into asparagine that served as an additional detoxification molecule. The constant Glu levels in the phloem sap suggested that Glu was continuously synthesized and supplied into the phloem regardless of leaf age. Further study using transgenic tobacco lines, harboring the promoter of the GLU1 gene (encoding Arabidopsis [Arabidopsis thaliana] Fd-GOGAT) fused to a GUS reporter gene, revealed that the expression of Fd-GOGAT remained higher in young leaves compared to old leaves, and higher in the veins compared to the mesophyll. Confocal laser-scanning microscopy localized the Fd-GOGAT protein to the phloem companion cells-sieve element complex in the leaf veins. The results are consistent with a role of Fd-GOGAT in supplying Glu for the synthesis and transport of amino acids. Taken together, the data provide evidence that the GS-GOGAT pathway and GDH play distinct roles in the source-sink nitrogen cycle of tobacco leaves.
利用[¹⁵N]铵和[2-¹⁵N]谷氨酸示踪剂,研究了烟草(Nicotiana tabacum L. cv Xanthi)幼叶和老叶中的谷氨酸(Glu)代谢及氨基酸转运。无论叶龄如何,[¹⁵N]铵的同化作用均通过谷氨酰胺合成酶(GS;EC 6.1.1.3)和谷氨酸合酶(铁氧还蛋白[Fd]-谷氨酰胺合成酶;EC 1.4.7.1;NADH-谷氨酰胺合成酶;EC 1.4.1.14)在光照和黑暗条件下进行,且不依赖于谷氨酸脱氢酶(GDH;EC 1.4.1.2)。[¹⁵N]铵和铵的积累模式支持了GDH在[2-¹⁵N]Glu脱氨以提供2-氧代戊二酸和[¹⁵N]铵过程中的作用。在黑暗中,过量的[¹⁵N]铵被整合到天冬酰胺中,天冬酰胺作为一种额外的解毒分子。韧皮部汁液中谷氨酸水平恒定,这表明无论叶龄如何,谷氨酸都在持续合成并供应到韧皮部。使用携带与GUS报告基因融合的GLU1基因(编码拟南芥[Arabidopsis thaliana]Fd-谷氨酰胺合成酶)启动子的转基因烟草品系进行的进一步研究表明,与老叶相比,Fd-谷氨酰胺合成酶在幼叶中的表达仍然更高,与叶肉相比,在叶脉中的表达更高。共聚焦激光扫描显微镜将Fd-谷氨酰胺合成酶蛋白定位到叶脉中的韧皮部伴胞-筛管分子复合体。这些结果与Fd-谷氨酰胺合成酶在为氨基酸合成和转运提供谷氨酸方面的作用一致。综上所述,这些数据提供了证据,表明GS-谷氨酰胺合成酶途径和GDH在烟草叶片的源-库氮循环中发挥着不同的作用。