Guescini M, Pierleoni R, Palma F, Zeppa S, Vallorani L, Potenza L, Sacconi C, Giomaro G, Stocchi V
Istituto di Chimica Biologica Giorgio Fornaini, Università degli Studi di Urbino, Via Saffi 2, 61029 Urbino (PU), Italy.
Mol Genet Genomics. 2003 Sep;269(6):807-16. doi: 10.1007/s00438-003-0894-3. Epub 2003 Aug 1.
The nitrate assimilation pathway represents a useful model system in which to study the contribution of a mycorrhizal fungus to the nitrogen nutrition of its host plant. In the present work we cloned and characterized the nitrate reductase gene (tbnr1) from Tuber borchii. The coding region of tbnr1 is 2,787 nt in length, and it encodes a protein of 929 amino acids. Biochemical and Northern-blot analyses revealed that nitrate assimilation in T. borchii is an inducible system that responds mainly to nitrate. Furthermore, we cloned a nitrate reductase cDNA (tpnr1) from Tilia platyphyllos to set up a quantitative real-time PCR assay that would allow us to determine the fungal contribution to nitrate assimilation in ectomycorrhizal tissue. Using this approach we demonstrated that the level of tbnr1 expression in ectomycorhizae is eight times higher than in free-living mycelia, whereas tpnr1 transcription was found to be down-regulated after the establishment of the symbiosis. Enzymatic assays showed that NADPH-dependent nitrite formation markedly increases in ectomycorrhizae. These findings imply that the fungal partner plays a fundamental role in nitrate assimilation by ectomycorrhizae. Amino acid determination by HPLC revealed higher levels of glutamate, glutamine and asparagine in symbiotic tissues compared with mycelial controls, thus suggesting that these amino acids may represent the compounds that serve to transfer nitrogen to the host plant.
硝酸盐同化途径是一个有用的模型系统，可用于研究菌根真菌对其宿主植物氮营养的贡献。在本研究中，我们克隆并鉴定了来自白块菌（Tuber borchii）的硝酸盐还原酶基因（tbnr1）。tbnr1的编码区长度为2787个核苷酸，编码一个由929个氨基酸组成的蛋白质。生化分析和Northern杂交分析表明，白块菌中的硝酸盐同化是一个主要对硝酸盐作出反应的诱导系统。此外，我们从阔叶椴（Tilia platyphyllos）中克隆了一个硝酸盐还原酶cDNA（tpnr1），以建立一种定量实时PCR检测方法，该方法可使我们确定真菌对外生菌根组织中硝酸盐同化的贡献。使用这种方法，我们证明外生菌根中tbnr1的表达水平比自由生活的菌丝体高八倍，而tpnr1的转录在共生建立后被下调。酶活性测定表明，外生菌根中依赖NADPH的亚硝酸盐形成明显增加。这些发现表明真菌伙伴在外生菌根的硝酸盐同化中起基本作用。通过HPLC测定氨基酸发现，与菌丝体对照相比，共生组织中谷氨酸、谷氨酰胺和天冬酰胺的水平更高，因此表明这些氨基酸可能是用于向宿主植物转移氮的化合物。
