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Plant and Cell Physiology Advance Access published online on March 8, 2006
Plant and Cell Physiology, doi:10.1093/pcp/pcj028
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Plant and Cell Physiology 2006 © The Japanese Society of Plant Physiologists (JSPP); all rights reserved.
Received January 25, 2006
Accepted March 2, 2006

Regular Paper

Phosphoenolpyruvate Carboxylase Plays a Crucial Role for Limiting Nitrogen Fixation in Lotus japonicus Nodules

Mika Nomura 1 *, Ha Thu Mai 1, Miho Fujii 1, Shingo Hata 2, Katsura Izui 2, and Shigeyuki Tajima 1

1 Faculty of Agriculture, Kagawa University, Miki, Kita, Kagawa, 761-0795 Japan
2 Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, 606-8502 Japan

* To whom correspondence should be addressed.
Mika Nomura, E-mail: nomura{at}ag.kagawa-u.ac.jp


  Abstract

Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) has believed to play a significant role for supporting nitrogen fixation via an anaplerotic CO2 fixation for recycling carbon in nodules. Using antisense technique, we diminished the expression levels of the nodule enhanced PEPC gene (Ljpepc1) in a determinate legume plant (Lotus japonicus) for surveying the influence to symbiotic phenotype and biochemical parameters.

Three independent transgenic L. japonicus plants (designated as Asppc1, Asppc2 and Asppc3) were prepared using Ljpepc1 DNA fragment which is under the control of cauliflower mosaic virus 35S promoter. Extensive suppression of Ljpepc1 transcript in nodules of Asppc plants (T3 homologous plants) was confirmed by RNA gel blot, western blot and the enzyme activity assays. In nodules of Asppc plants, PEPC activity reduced to ca. 10% of non-transformants and the plants showed a typical nitrogen deficient symptom without nitrogen nutrient supply, and returned to normal growth with supply of 2.5 mM nitrate. Acetylene reduction activities per fresh weight of nodules of these Asppc plants decreased by 29% in 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 asparagines aminotransferase activities was observed in Asppc nodules. In addition, sucrose, succinate, asparagine, asparatate and glutamate contents also decreased in Asppc nodules. The data was discussed on a role of PEPC for the carbon/nitrogen metabolic flux in nodules.

Keywords: Carbon; Lotus japonicus; Nitrogen; Nodule; Phosphoenolpyruvate carboxylase.
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