Function Analyses Of Low-Affinity Nitrate Transporter Gene OsNPF2.4 In Rice

Rice is one of the most important crops in the world, accounting for 1/5 of the global crop acreage. The annual production is about 480 million tons,1/4 of total grain output in the world. There is more than 1/2 of the world population with rice as the staple food. Meanwhile, rice is also one of the most important crops in China. Due to the small genome size, the rice is used as an important model plant for monocot species, and has abundant and profound genomic research foundation. Nitrogen （N） is the most critical limiting factor for crop production. Nitrate （NO₃^-） and ammonium （NH₄⁺） are the most important inorganic N absorbed by plants. Rice is commonly grown in flooded field, however, the aerenchyma cells in the rice root can transport oxygen from the shoot to the root then release to the rhizosphere, immediately, the nitrification starts and ammonium converts to nitrate.The highest nitrate concentration in the flooded field can up to 1.72±0.42 mM （Eguchi et al., 2009）. Moreover, the intermittent irrigation at later growth and developmental stages can greatly increase the NO₃^- content in rhizosphere. Therefore, even in the flooded field, up to 40% of the total N absorbed by rice roots is in the form of nitrate, and at sometimes the amount of nitrate absorbed by rice roots may be comparable with the amount of ammonium at sometimes.To acquire enough N for growth, the plants have to cope with the fluctuation in the availability of N sources in the soil in temporal and spatial. Therefore, the plants have developed two nitrate uptake systems, the low-affinity nitrate transport system （LATS） and high-affinity nitrate transport system （HATS）.The two gene families of nitrate transporters, NRT1/PTR （NPF） and NRT2, are responsible for the LATS and HATS,respectively. Compared with the NRT2 in rice, the research of NRT1/PTR （NPF） in terms of nitrate utilization is very rare. In this thesis, we focus on a low-affinity nitrate transport gene OsNPF2.4 in rice. qRT-PCR> GUS and eGFP reporter genes were applied to analyze the expression pattern in rice. Xenopus laevis Oocytes heterologous expression system and two-electrode voltage clamp analysis was performed to analyze the substrate uptake property; RT-PCR,qRT-PCR and Southern blot were employed to identify the OsNPF2.4 T-DNA inserational mutants and overexpression lines and analyze the the expression of relative genes; The N uptake and redistribution of OsNPF2.4 knockout mutants were analyzed by 15N labeled NO₃^-; The yield and agronomic traits of OsNPF2.4 knockout mutants and overexpression lines grown in a paddy soil with alternative dry-flooding irrigation were also analysed. The main results were listed as follows:1. Bioinformatic and qRT-PCR analysis showed that the open reading frame of OsNPF2.4 is composed of two introns and three exons, encoding a protein composed of 583 amino acids. OsNPF2.4 is localized on plasma membrane and has 12 transmembranes （TMs）. In root, the expression of OsNPF2.4 could be enhanced by supply of high NO₃^- （2.5 mM） and suppressed by K+ deficiency. In shoot,OsNPF2.4 was expressed most abundantly in high NO₃^- supplied youngest leaf blade. N starvation could enhance the expression of OsNPF2.4 in older （3rd-5th） leaf blade and sheath. In addition, K+ deficiency down-regulated expression of OsNPF2.4 in culm and leaf sheath, but up-reregulated its expression in leaf blade.2. Spatial expression pattern of OsNPF2.4 was detected in promoter-GUS transgenic rice in different tissues and different growth stage.OsNPF2.4 is expressed throughout the entire root except the tip, vascular of the root-shoot junction, leaf sheath and blade, anther, germinated seed and seed hull. Cross-sections analysis showed that OsNPF2.4 is mainly expressed in root epidermis, xylem parenchyma and phloem company cells, and leaf phloem cells.3. Xenopus laevis Oocytes heterologous expression system and two-electrode voltage clamp analysis showed that OsNPF2.4 is a pH dependent low affinity NO₃^- transporter, it showed a NO₃^- uptake acticty at pH5.5, and lost the NO₃^- uptake acticty at pH7.4. OsNPF2.4 cannot efflux NO₃^- from the cell. Two-electrode voltage clamp analysis indicates that OsNPF2.4 has no peptide transport activity.4. Knockout of OsNPF2.4 seriously decreased the NO₃^- absorb from medium, and overexpresson of OsNPF2.4 can increase the NO₃^- absorption from medium. Short-term NO₃^- acquisition was analyzed by exposing the roots to 0.25 mM or 2.5 mM 15NO₃^- for 10 min. Compared with WT, OsNPF2.4 konckout mutants showed significant lower 2.5 mM 15NO₃^- influx rate into roots, while no difference of 0.25 mM 15NO₃^- influx rate was observed between them. In long-term treatments, knockout mutation of OsNPF2.4, however, decreased total NO₃^- uptake at both lower （0.25 mM） and higher （2.5 mM） NO₃^- levels, although the difference of both NO₃^- concentrations and total N per plant between the mutants and WT was larger at higher NO₃^- level. We found that the major components for high affinity NO₃^- transport system （HATS）, both OsNAR2.1 and OsNRT2.1 were down-regulated in the low NO₃^- supplied OsNPF2.4 konckout mutants than WT. It is interesting that increase of NO₃^- supply from 0.25 mM to 2.5 mM increased total N uptake by 20-35% in the root, culms and sheath, and blade of WT, however, it increased total N uptake in the mutants slightly.5.15NO₃^- redistribution from the oldest leaf to other organs analysis showed that concentration of 15N distributed into root, culm and sheath, youngest （1st） leaf blade, and total amount of I5N accumulated in these organs of the mutants were only about 50%-60% of WT. Moreover, the relative re-distribution ratio of 15N was significantly decreased in the root, but increased in culm and youngest （1st） leaf blade of the mutants in comparison to WT.6. Xylem sap of OsNPF2.4 konckout mutants and WT supplied with 0.25 mM or 2.5 mM NO₃^- was collected, and NO₃^- and K+ content was analyzed. Both NO₃^- and K+concentrations in the xylem sap between WT and osnfp2.4 mutants were similar at 0.25 mM NO₃^- condition, however, they showed about 42% and 27% lower, respectively, in the mutants than in WT at 2.5 mM NO₃^- condition when temporary removed both NO₃^- and K+ in the culture solution for 2 and 4 h before sampling the xylem sap. Interestingly, NO₃^-transport rate in the xylem of the mutants was only about 45% of that in WT at 2 h, while it did not show any difference at 4 h. At meanwhile, K transport rate in the xylem of the mutants was also significantly lower than that of WT. Moreover, xylem sap K+ in osnpf2.4 mutants was further decreased as the time extending.OsNPF2.4 knockout decreased K+ concentration in root, culm and sheath, but increased K+ distribution in shoot.Taken together, OsNPF2.4 is a pH dependent low-affinity NO₃^-transporter, and mainly responsible for nitrate uptake and redistribution at high NO₃^- condition. Moreover, disruption of OsNPF2.4 affects the shuttle of NO₃^- and K+ in rice.