Adaptation To Pi Deficiency And Effects To Mycorrhiza Of The Rice Plasma Memberane H~+-Atpase Genes

Phosphorus (P) is essential for plant growth and development due to its involvement in the processes of energy metabolism and synthesis of nucleic acids and membranes. However, the low availability of soil P is a major constraint for crop production in many agricultural systems worldwide. Higher plants thus alter their architecture and metabolism to acquire sparingly soluble P from soil. It was reported that plants are able to mobilize P by acidification of the rhizosphere by release of H+ from the roots to balance excess intake of cations over anions. Pi uptake into the root symplasm involves transport from the apoplast where Pi concentration is less than 2μM, across the plasma membrane (PM), and to the cytosol where Pi concentration ranges from 5-17 mM. The negative membrane potential and the large difference between external and internal Pi concentration necessitate that a steep electrochemical gradient must be overcomed for Pi transport into root cells, thus a high-affinity, energy-consuming transport mechanism driven by PM H+-ATPase is required. The mechanism underlying H+ release is a primary process caused by H+-ATPase activity in the plasmalemma of root cells. This enzyme acts as a primary transporter by pumping protons out of the cell, thereby creating pH and electric potential differences across the PM. Rice (Oryza sativa), an economically important crop and a monocot model plant for scientific research, possesses ten PM H+-ATPase genes. Consequently, research on functional characterization of rice PM H+-ATPase genes with regard to rice nutrients uptake and translocation is of extreme importance for understanding of the mechanism and germplasm enhancement.The studies on the adaptation of plant PM H+-ATPases to Pi deficiency were all carried out by dicotyledonous plants, while little is known about that of the monocotyledonous plants. In the present work, the adaptation of rice root PM H+-ATPase activity to Pi deficiency was studied. Rice plants (Oryza sativa cv. Nipponbare L.) were fed with or without phosphate in hydroponics culture experiment. At the seedling stage the PM of rice roots was isolated by two phase system. The PM H+-ATPase hydrolytic activity was analyzed for elucidating the response of the PM H+-ATPase of rice root to Pi deficiency. Moreover, tissue localization analysis for rice H+-ATPase genes was performed using transgenic plants carrying promoter::GUS (β-Glucuronidase) reporter fusions. Furthermore, with wild type (WT) rice (Oryze Sativa ssp. Japonica cv. Hitomebore) as control, we investigated the physiological function of OsA8, a member of rice PM H+-ATPase gene family, by comparison of the responses between the Tos17 insertional homozygous mutant of OsA8 gene and its WT in nutrient uptake and translocation under arbuscular mycorrhizal (AM) colonization. The main results are shown as follow:1. The adaptation of PM H+-ATPase of rice roots to Pi deficiency is supported by the following results obtained in vitro. Compared with roots from Pi-sufficient plants, the Pi-deficient rice roots had higher hydrolytic and pumping activity of PM H+-ATPase as well as Vmax. The higher activity of H+-ATPase of rice roots under Pi deficiency was mirrored either by a higher expression rate (transcriptional level) of genes encoding PM H+-ATPase or an enhanced synthesis rate of the enzyme proteins. Higher H+ pumping activity than hydrolytic activity, and a higher Vmax, with a lower Km of PM H+-ATPase data suggest that the enhancement of PM H+-ATPase activity by Pi deficiency in rice roots could also be regulated at post-translational level.2. Multiple sequence alignment and phylogenetic tree analysis showed that the ten rice H+-ATPase genes, which could be grouped into five subfamilies, are very closely related with sequence identities over 80%. This indicates that these genes might have evolved through gene duplication events during rice evolution.Transgenic rice plants carrying promoter::GUS fusions were used for the determination of spatial expression pattern and promoter strength of OsATPase genes (OsAs). The expression of OsA2, OsA3, OsA4 and OsA7 was detected in the root tips, lateral roots, root-shoot junction and leaves under normal conditions. While the expression of OsA8 was barely detectable under normal conditions. A possible explanation for this could be that its expression is restricted to particular cell types.3. In contrast to histochemical analysis of GUS activity driven by other promoter fragments of AM-induced genes of solanaceous species or rice. We did not observe any reporter gene activity in any tissues with the OsA8 promoter under AM colonization.4. OsA8 knockout mutants showed a lower colonization rate and higher Pi concentration in roots compared with WT plants. Interestingly, expression of genes of H+-ATPase family other than OsA8 and genes belonging to the Phtl family showed similar expression patterns, namely mycorrhizal symbiosis decreased of the OsAl, OsA2, OsA3, OsA7, OsA9 and Phtl family gene (except OsPT8) expression in osa8 mutant root.5. Two independent transgenic lines with enhanced OsA8 expression showed increased activity of PM H+-ATPase under normal condition. Pi uptake and particularly the translocation of P from roots to shoots were also enhanced. These results are consistent with the enhanced expression of genes belonging to PM H+-ATPase and Phtl family. OsA8 over-expression decreased OsAl, OsA3 and OsA9 of H+-ATPase family and OsPTl, OsPT3, OsPT4 and OsPT9 of Phtl family.The results indicated that OsA8 played very important roles in the inoculation of rice roots with AMF and uptake and translocation of Pi, as well as in regulation of root growth in the rice. Under Pi deficieny and AMF inoculation, Pi uptake and translocation was changed by OsA8 gene affected PM H+-ATPase activityExpression of many other Pi transporters was regulated by changed Pi concentration in the OsA8 gene knockout or over-expression rice roots and shoots. It was concluded that maintenance of OsA8 gene’s expression is critical to keep normal AMF inoculation and Pi uptake and translocation in addition to normal root morphology of rice plants.