Plant Biomass and Nutrition, Institute of Botany, Darmstadt University of Technology, Darmstadt, Germany.
PLoS One. 2010 Jan 27;5(1):e8917. doi: 10.1371/journal.pone.0008917.
Glutamine is one of the primary amino acids in nitrogen assimilation and often the most abundant amino acid in plant roots. To monitor this important metabolite, a novel genetically encoded fluorescent FRET-reporter was constructed and expressed in Arabidopsis thaliana. As a candidate for the glutamine fluxes, the root tip localized, putative amino acid transporter CAT8 was analyzed and heterologously expressed in yeast and oocytes.
Rapid and reversible in vivo fluorescence changes were observed in reporter-expressing root tips upon exposure and removal of glutamine. FRET changes were detected at acid and neutral pH and in the presence of a protonophore, suggesting that part of the glutamine fluxes were independent of the pH. The putative amino acid transporter CAT8 transported glutamine, had a half maximal activity at approximately 100 microM and the transport was independent of external pH. CAT8 localized not only to the plasma membrane, but additionally to the tonoplast, when tagged with GFP. Ultrastructural analysis confirmed this dual localization and additionally identified CAT8 in membranes of autophagosomes. Loss-of function of CAT8 did not affect growth in various conditions, but over-expressor plants had increased sensitivity to a structural substrate analog, the glutamine synthetase inhibitor L-methionine sulfoximine.
The combined data suggest that proton-independent glutamine facilitators exist in root tips.
谷氨酰胺是氮同化过程中的主要氨基酸之一，也是植物根系中最丰富的氨基酸。为了监测这种重要的代谢物，构建并在拟南芥中表达了一种新型的遗传编码荧光 FRET 报告器。作为谷氨酰胺通量的候选物，对定位于根尖的假定氨基酸转运蛋白 CAT8 进行了分析，并在酵母和卵母细胞中异源表达。
在暴露和去除谷氨酰胺时，报告基因表达的根尖快速且可逆地发生体内荧光变化。在酸性和中性 pH 值以及质子载体存在的情况下检测到 FRET 变化，表明部分谷氨酰胺通量与 pH 值无关。假定的氨基酸转运蛋白 CAT8 转运谷氨酰胺，其半最大活性约为 100μM，且该转运独立于外部 pH 值。CAT8 不仅定位于质膜，还定位于 GFP 标记的液泡膜。超微结构分析证实了这种双重定位，并在自噬体的膜中鉴定出 CAT8。CAT8 的功能丧失并不影响各种条件下的生长，但过表达植物对结构类似物，即谷氨酰胺合成酶抑制剂 L-甲硫氨酸亚砜胺的敏感性增加。
综合数据表明，根尖存在质子非依赖型谷氨酰胺促进剂。
