Identification of a Sed5-like SNARE gene LjSYP32-1 that contributes to nodule tissue formation of Lotus japonicus.

We identified a Sed5-like clone LjSYP32-1 which contributes to nodule tissue formation and plant growth in Lotus japonicus. In the L. japonicus expressed sequence tag (EST) clone databases of Kazusa DNA Research Institute, another syntaxin-related clone (LjSYP32-2) was also detected, and the nucleotide and amino acid sequences of these two clone are very similar to each other. Real-time PCR and promoter analysis indicated that expression of LjSYP32-1 was dominant compared with LjSYP32-2 in the various plant organs. Promoter analysis and in situ hybridization revealed that LjSYP32-1 was expressed significantly in the inner cortex cell layer surrounding the infected zone of young nodules and in the meristem area of developing lateral root. To explore the function and physiological role of LjSYP32-1 in nodules and other plant organs, stable transformation lines of L. japonicus expressing either sense or antisense LjSYP32-1 were prepared. The antisense plants showed a significantly retarded plant growth phenotype, suggesting a role for LjSYP32-1 in supporting plant growth. In the same transgenic lines, the plants were capable of forming nodules, but the acetylene reduction activity was reduced by around 50% per plant. The nodules were much smaller and some nodules were fused to each other by sharing the inner cortex. The rate of occurrence of such irregular nodules was twice that observed in wild-type plants. The data suggest that LjSYP32-1 contributes to the support of plant growth and normal nodule tissue differentiation.

Phosphoenolpyruvate carboxylase plays a crucial role in limiting nitrogen fixation in Lotus japonicus nodules.

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.