Nomura Mika, Mai Ha Thu, Fujii Miho, Hata Shingo, Izui Katsura, Tajima Shigeyuki
Faculty of Agriculture, Kagawa University, Miki, Kita, Kagawa, 761-0795 Japan.
Plant Cell Physiol. 2006 May;47(5):613-21. doi: 10.1093/pcp/pcj028. Epub 2006 Mar 8.
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.
磷酸烯醇式丙酮酸羧化酶（PEPC，EC 4.1.1.31）被认为通过回补性二氧化碳固定在支持根瘤中碳循环的固氮过程中发挥重要作用。利用反义技术，我们降低了有限生长型豆科植物（百脉根）中根瘤增强型PEPC基因（Ljpepc1）的表达水平，以研究其对共生表型和生化参数的影响。使用受花椰菜花叶病毒35S启动子控制的Ljpepc1 DNA片段制备了三株独立的转基因百脉根植株（分别命名为Asppc1、Asppc2和Asppc3）。通过RNA凝胶印迹、蛋白质免疫印迹和酶活性测定，证实了Asppc植株（T3同源植株）根瘤中Ljpepc1转录本受到广泛抑制。在Asppc植株的根瘤中，PEPC活性降低至非转化植株的约10%，并且在不供应氮营养时植株表现出典型的缺氮症状，而当供应2.5 mM硝酸盐时恢复正常生长。这些Asppc植株根瘤鲜重的乙炔还原活性在接种后35天（dai）时下降了29%。在接种后35天，使用Asppc植株对各种酶活性和代谢物水平进行了检测。在Asppc根瘤中观察到蔗糖合酶和天冬酰胺转氨酶活性显著降低。此外，Asppc根瘤中的蔗糖、琥珀酸、天冬酰胺、天冬氨酸和谷氨酸含量也有所下降。本文根据PEPC在根瘤碳/氮代谢通量中的作用对这些数据进行了讨论。
