Phosphoenolpyruvate carboxylase plays a crucial role in limiting nitrogen fixation in Lotus japonicus nodules
Phosphoenolpyruvate carboxylase plays a crucial role in limiting nitrogen fixation in Lotus japonicus nodules
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DOI：
10.1093/pcp/pcj028
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发表时间：
2006-05-01
影响因子：
4.9
通讯作者：
Tajima, S
中科院分区：
文献类型：
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作者：
Nomura, M;Mai, HT;Tajima, S
磷酸烯醇式丙酮酸羧化酶(PEPC，EC 4.1.1.31)被认为在支持固氮中发挥重要作用，它通过逆行CO2固定来循环根瘤中的碳。我们利用反义技术降低了结瘤增强型PEPC基因(LjPepc1)在豆科植物莲花中的表达水平，以观察共生表型和生化参数的影响。利用花椰菜花叶病毒35S启动子控制下的LjPepc1 DNA片段，获得了3株独立的转基因植株(分别命名为Asppc1、Asppc2和Asppc3)。RNA凝胶印迹、Western印迹和酶活性分析证实，LjPepc1转录本在Asppc植株的根瘤中被广泛抑制)。在Asppc植株的根瘤中，PEPC活性下降到非转化体的10%左右，植株在没有氮素供应的情况下表现出典型的缺氮症状，当硝酸盐供应到2.5 mM时，植株恢复正常生长。在侵染后35d，这些Asppc植株的单位鲜重的乙炔还原活性下降了29%。利用35DAI的Asppc植物对各种酶活性和代谢产物水平进行了测定。Asppc根瘤中蔗糖合成酶和天冬氨酸氨基转移酶活性显著降低。此外，Asppc根瘤中蔗糖、琥珀酸、天冬酰胺、天冬氨酸和谷氨酸的含量也有所下降。从PEPC在结核中碳/氮代谢通量中的作用的角度讨论了这些数据。
Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) is believed to play a significant role in supporting nitrogen fixation via anaplerotic CO2 fixation for recycling carbon in nodules. Using the antisense technique, we decreased the expression levels of the nodule-enhanced PEPC gene (Ljpepc1) in a determinate legume plant (Lotus japonicus) in order to look at the influence of the symbiotic phenotype and biochemical parameters. Three independent transgenic L. japonicus plants (designated as Asppc1, Asppc2 and Asppc3) were prepared using a Ljpepc1 DNA fragment which is under the control of the cauliflower mosaic virus 35S promoter. Extensive suppression of the Ljpepc1 transcript in nodules of Asppc plants T3 homologous plants) was confirmed by RNA gel blot, Western blot and enzyme activity assays. In nodules of Asppc plants, PEPC activity was reduced to about 10% of that of non-transformants and the plants showed typical nitrogen-deficient symptoms without a supply of nitrogen nutrient, and returned to normal growth when nitrate was supplied at 2.5 mM. The acetylene reduction activity per fresh weight of nodules of these Asppc plants decreased by 29% at 35 dai (days after infection). Various enzyme activities and metabolite levels were surveyed using Asppc plants at 35 dai. Significant reduction of sucrose synthase and asparagine aminotransferase activities was observed in Asppc nodules. In addition, sucrose, succinate, asparagine, aspartate and glutamate contents also decreased in Asppc nodules. The data are discussed in terms of a role for PEPC in the carbon/nitrogen metabolic flux in nodules.