Functional Analysis Of A Plasma Membrane Proton Pump Gene, OsA8, In Response To Nitrogen,Phosphorus And Potassium In Rice

Advancements in molecule biology provide plentiful materials and effective methods for researches in field of plant nutrition. It is therefore promising to reveal the molecule mechanism of nutrient uptake and translocation in plants. Based on the theory of chemical osmosis and experimental analyses in translocation of ions across plasma membrane and electrical physiology in last two decades, elementary mechanism of nutrient across plasma membrane has been elucidated. Due to coupling of proton motive force resulting from proton transport across membrane with the active transport of soluble matter and hormone, increasing interest is raised to the functions of H+-ATPases in nutrition uptake, translocation and redistribution, as well as growth and development. Although there are many numbers in the large H+-ATPases family, few of them have been characterized for their physiological functions. Particularly, no plasma membrane H+-ATPase gene associated with nutrient acquisition has been reported in rice so far. Nitrogen (N), phosphorus (P) and potassium (K) are three macro essential nutrients of plants and are often insufficient in many soils. Due to the low avaiability of P in nature soils, comprehensive attention has been paid to the physiological and molecular mechanisms of plants to adapt the deficiency of P. Research in soybean has shown that entire activity of plasma membrane ATPase is associated with P uptake and translocation. Rice (Oryza Sativa) is a model plant and an economically important crop, there are total10members of OsAs, plasma membrane proton ATPase genes in rice. Researches on function of ATPase in rice relating to its nutrient uptake and translocation is, therefore, extremely important for improving the plant nutrient use efficiency by molecular approaches.In this study, using wild type rice (Oryzae Sativa ssp. Japonica cv. Hitomebore) as a control, we investigated the physiological functions of OsA8, a plasma membrane proton-ATAase by comparison of the responses between Tos17insertional homozygote mutant of OsA8gene and its wild type in nutrient uptake and translocation. The main results are shown as follows:1. OsA8is expressed in leaves under normal nutrient supply condition and roots in P deficiency of rice, but its expression level is low. It is worth noting that contrary to the report in soybean that P deficiency enhanced ATPase activity, we observed that expression of OsA8was repressed in leaves and enhanced in roots by P deficiency in rice plant. Deficiency of K and N also decreased the expression of OsA8, but the effect was much weaker in comparison with that of P deficiency.2. We obtained the Tos17insertional OsA8mutant, H0310, from the Rice Genome resource center, National Institute of Agrobiological Science (Japan). Using suppression PCR and IR-PCR methods, we identified14OsA8homozygotes. RT-PCR analysis confirmed that no detectable expression of OsA8in these mutants.3. Knockout of OsA8reduced biomass of roots and aerial part in addition to affect root morphology. Under hydroponic growth condition, either with supply of normal nutrient solution or with P deficient solution, the mutant showed reduced total length, volume, surface area and number of root tips. However, the mean diameter of roots grown in normal nutrient solution was not affected although it was affected under P deficient condition. The mutant also showed less number of tillers and retardation of tillering. No obvious effect was observed on plant height.4. Consistent with its expression pattern, we found that OsA8is linked to P uptake, particularly to the translocation of P from roots to shoots. Under P deficient condition, root P concentration of the mutant was significantly higher than that of wild type. However, no obvious difference of root P concentration was observed under normal nutrient supply condition. Under normal nutrient condition, shoot P concentration of the mutant was significantly lower than that of wild type, no obvious difference could be found in P deficient condition. These suggest that, compared with control, P translocation from root to shoot of OsA8mutant was obvious depressed.5. Similar to its response to P deficiency, OsA8responded to NO3--N and K deficiency. However, the extent of the responses was much smaller. Different N and K treatments exert no obvious influence on morphology of the mutant. However, the uptake of K and NO3--N was repressed in the mutant whereas their translocation was improved. 6. In addition to the influences on uptake and translocation of NO3--N, P and K, knockout of the gene increased concerntration of soluble sugar in roots of the mutant. Consistently, activities of H+-ATPase and root reductase were also enhanced in the mutant. Contrast with lower biomass, suggest that higher energy expense also can’t compensate fully the influence of knock out of OsA8.7. RT-PCR analyses showed that Knockout of OsA8gene affected expression of several OsA and OsPhtl genes. Knockout of OsA8increased the expression of OsAl and OsA2in the roots of Pi-starved mutants. However, a lower abundance of OsA2was noticed in leaves of the mutant. In addition, OsA6appears to be induced only in roots of Pi-sufficient wild type plants. This suggests a complex regulation of expression of the members of the OsA family in roots and leaves under Pi starvation conditions. In addition, P-starvation strongly enhanced expression of OsPT6, a member of phosphate transporters in Phtl family in its wild type in the cultivar Hitomebore, however, did not induce the expression of OsPT6in roots of the mutant. Knockout of OsA8did not noticeably affect the expression of other members of the Phtl family.In summary, we found that function of the plasma membrane proton pump OsA8was linked to phoshphorus uptake, translocation of NO3--N and potassium in rice. Knockout of OsA8influenced the expression of some genes in the Phtl family besides H+-ATPase activities, root reductase activities, and the concentration of soluble sugar in root. Maintenance of the gene’s expression is important to keep normal nutrient uptake and translocation in addition to normal growth of rice.