Extracellular ATP, apyrase and nodulation of non-legumes

Abstract

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] ATP is a universal energy source for biochemical reactions in all living organisms. Extracellular ATP (eATP) is well studied in animal processes such as neurotransmission, platelet aggregation, and muscle function. In plants, eATP is related to the generation of ROS (reactive oxygen species), cytoplasmic calcium elevation, drought response and cell growth. Although previous work had shown that the addition of ATP to plants could elicit a cellular response, the actual production of extacellular ATP by plants had not been documented. Extracellular ATP (eATP) on the surface of legume roots was visualized through the use of a novel reporter protein consisting of a cellulose binding domain fused to the luciferase enzyme. This fusion protein bound to the root surface via the cellulose binding domain and served as a reporter for the presence of eATP by light production from the luciferase in the presence of the substrate luciferin. This reporter provided a means to monitor eATP in the living root. Using this reporter system, eATP was highly localized to the root hair tips, where the cell is actively growing. High levels of eATP also coincided to other regions of the root undergoing active growth. The release of eATP was dependent on calcium and was reduced by inhibition of vesicular trafficking. The latter results suggtests that eATP exists the plant cell via vesicular exocytosis, similar to the method of release documented in animal cells. ROS is important for root hair growth and is also highly expressed at the root hair tip. Addition of eATP triggered a strong ROS response at the root hair tip, which was significantly reduced upon the addition of apyrase. Previous research had implicated the soybean GS52 ectoapyrase in the mechanism of rhizobial infection of root hairs. An ectoapyrase's catalytic domain is extracellular consistent with a possible role in hydrolyzing eATP released by the plant. To determine the role of apyrase in soybean, ecto-apyrase (GS52) was silenced by Agrobacterium rhizogenes mediated hairy root transformation. GS52 silenced soybean roots showed significantly reduced nodule numbers. This reduction in nodule numbers could be reversed by the addition of exogenous ADP. The data suggest that the GS52 ectoapyrase plays a critical role in soybean nodulation, perhaps by modulating the levels of eATP. Indeed, the addition of exogenous ATP significantly reduced nodule formation. An important long term goal of the nodulation research community is to transfer nodule formation from legumes to non-legumes, which do not normally interact with rhizobia. Nodule formation is initiated by recognition by the plant of lipo-chitin nodulation factors (Nod factor) produced by the rhizobium. The plant receptors for the Nod factor were recently idenfied (e.g., LjNFR1 and LjNFR5 from Lotus japonicus). Preliminary results indicate that the transfer of the genes for LjNFR1 and LjNFR5 to some cultivars of tomato resulted in the formation of nodule-like structures when inoculated with rhizobia. In some cases, bacteria could be visualized withint these nodule-like structures and could be isolated. These results, if confirmed, may provide the initial, critical steps toward the ultimate goal of creating nodulating, non-leguminous crop plants.