| Diphosphonucleotide phosphatase(PPD) is a subfamily of purple acid phosphatase(PAP). As IA257 is a novel symbiosis-specific expressed gene isolated from A. sinicus by suppressive subtractive hybtidization in our previous work. Nucleotides sequence and amino acids sequence of As IA257 are similar to PPD1 from yellow lupin. Therefore, As IA257 is named As PPD1.To address the function and mechanism of As PPD1 in legume-rhizobium symbiosis, its expression patterns, enzyme activity, cellular localization, and phenotypes associated with its over-expression and RNA interference(RNAi) were investigated. The results can be summarized as follows.The expression of As PPD1 was up-regulated in roots and nodules after inoculation with rhizobia. Phosphate starvation reduced the levels of As PPD1 transcripts in roots while increased those levels in nodules. We confirmed the acid phosphatase and phosphodiesterase activities of recombinant As PPD1 purified from Pichia pastoris, and demonstrated its ability to hydrolyze ADP and ATP in vitro. Cellular localization showed that As PPD1 located on the plasma membranes in hairy roots and on the symbiosomes membranes in root nodules. Over-expression of As PPD1 in hairy roots inhibited nodulation, while its silencing resulted in nodules early senescence and significantly decreased nitrogenase activity. Furthermore, HPLC measurement showed that As PPD1 silencing affects the ratio of ATP/ADP and the energy charge in nodules, and quantitative observation demonstrated the changes of As PPD1 transcripts level affected nodule primordia formation. Taken together, it is speculated that As PPD1 contributes to symbiotic ADP levels and energy charge control, and this is required for effective nodule organogenesis and nitrogen fixation.These results suggest that symbiosis-specific expressed of As PPD1 has regulatory function in symbiotic nitrogen fixation, which provides good experimental basis for further study regulatory mechanism. |
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