Characterization Of The Effect Of Phosphate Transporters OsPT5/OsPT7 And Proton-Pyrophosphatase AVP1D On Plant Phosphate Uptake And Development

Phosphorus (P) is an essential macronutrient for plant growth and development. It plays key roles in the processes of plant metabolic, including energy transfer, signal transduction, biosynthesis of macromolecules. However, one of the major causes in limiting crop production is the low availability of soil P due to inorganic fixation and formation of organic complexes. Plants have evolved several strategies to improve P absorption and utilization, including alterations in root morphology, modification of soil chemical properties around the roots, activation of high-affinity Pi transporters and activation of auxin transporters.Specific transport systems are essential for the uptake of Pi and for its translocation within plants. Pi transport across plant membranes is mediated by a number of families of transporters. In rice genome, there are a total of 13 genes encoding proteins that belong to the Phtl (Phosphate transporter 1) family, yet some of which have not been functionally characterized. In addition, to adapt Pi deficiency, plants alter their root architectureand enhance acidification of the rhizosphere by excretion of H+ from roots. However, the molecular regulatory mechanisms are still not clear.In the present work, we generated and functionally characterized the transgenic lines of overexpression of two members of rice Phtl family, OsPT5 and OsPT7, GFP translational fusion and mutants of Arabidopsis Auxin transporters (A UX/PIN/ABCB-FP) and the overexpression of AVP1D in tomato. Semi-quantitative (sq) RT-PCR, quantitative (q) RT-PCR, heterologous expression system, genetic engineering, confocal laser scanning microscope (CLSM) and isotope tracer techniques were used in the research. The main results are summarized as following:1. Bioinformatics analysis showed that OsPT5 and OsPT7 are present as single copy inthe genome of rice (cv. Nipponbare). Both genes are composed of a single exon. OsPT5 is localized on chromosome 4, and OsPT7 is localized on chromosome 3. Both qRT-PCR and sqRT-PCR showed that the expression of OsPT5 and OsPT7 was increased in roots and shoot upon Pi starvation. In general, the transcript abundance of OsPT5 was lower than that of OsPT7. The expression level of OsPT5 in roots is lower than that in shoots, while a opposite trend was observed for OsPT7. Yeast heterologous expression system suggested a Pi-affinity value (Km) of 65.7μM and 74μM, respectively, for OsPT5 and OsPT7, indicating that both OsPT5 and OsPT7 might be high affinity phosphate transporters.2. We successfully obtained the transgenic lines overexpressing OsPT5 and OsPT7. In a hydroponic culture system, both OsPT5 and OsPT7 overexpression lines showed sensitivity to P deficiency, enhanced biomass, and root/shoot ratio compared with wild type (WT) plants. Soluble and total P contents were increased in roots of OsPT5 overexpression lines, while were increased in both shoots and roots of OsPT7 overexpression lines. These results indicated that OsPT5 functions in P uptake, while in root and OsPT7 is responsible for not only P acquisition, but also P translocation to shoots. We also carried out a pot experiment with different P regimes. OsPT5 and OsPT7 overexpression lines displayed better growth performance under all P regimes, especially when supplied with low and intermediate levels of P. The total P concentration in unfilled rice hull and leaf blade, as well as seed-setting rate of OsPT5 overexpressing lines were significantly increased as compared with that of WT plants under low and intermediate Pi supply. Similarly, the total P concentration in unfilled rice hull, leaf and root were increased as compared with that of WT plants under low and intermediate Pi supplies. These results indicated OsPT5 might be involved in P translocation from old leaf to young leaf, and from leaf and panicle axis to the hull, while OsPT7 could play a role in both P acquisition and P mobilization from root to shoot.3. Transgenic tomato plants overexpressing AVP1D exhibited increased root-ward auxin transport and root acidification, and produced 25%(P=0.001) more marketable ripened fruits per plant under P-deficient conditions as compared with WT plants. Moreover, under low phosphate conditions, AVP1D overexpressing plants also displayed increased phosphate transport from leaf (source) to fruit (sink) comparing with WT plants.These results suggest that selection of tomato cultivars with increased proton pyrophosphatase gene expression could be a useful strategy for cultivating them in Pi deficiencies soils.4. Low P inhibits Arabidopsis primary root growth and promotes lateral root initiation and development. Our results demonstrated that decreased auxin levels in the root tip especially in the region surrounding the quiescent center were consistent with the primary root inhibition, while unchanged auxin levels in the epidermis and pericycle suggested enhanced auxin transport to these cell layers, which was consistent with enhanced lateral root initiation and development. In response to low P, the transcript abundance of ABCB19, PIN1, PIN2 and AUX1 was increased to facilitate auxin reflux at the root tip which resulted in primary root inhibition and promotion of lateral root development.