Function Analyses Of Ammonium Transporter Genes OsAMT1.1 And OsAMT2.1 In Rice

Rice(Oryza sativa L.)is one of the world's three major food crops that provide food for more than half of the world's population,accounting for 1/3 of the global cereal crop acreage.Nitrogen(N)is one of the major factors limiting crop growth and yield among the necessary nutrient elements for plant.In anaerobic paddy or acidic soils,the majority of inorganic N form is NH4+.To meet the different NH4+ concentration in the soil,higher plants have low-affinity ammonium transport system(LATS)and high-affinity ammonium transport system(HATS)to uptake NH4+.So far,two gene families have been identified:the AMT1 and AMT2 families involved in the low-affinity ammonium transport system(LATS)and high-affinity ammonium transport system(HATS),respectively.In rice,there are at least 10 putative OsAMT-like genes,which group into four sub-families(AMT1-AMT4)with one to three gene members.But the research of ammonium transporters in rice is still focus on the three high-affinity ammonium transporter genes.So far,there has been no report with mutants to analyses the function of ammonium transporter in rice.In this thesis,we focus on two putative ammonium transporter genes OsAMT1.1 and OsAMT2.1 in rice.QRT-PCR,GUS and eGFP reporter genes were applied to analyze the expression pattern of OsAMT1.1 in rice.Effects of OsAMT1.1 gene disruption on short-term ammonium influx rate in rice were analyzed by 15N labeled 15NH4+.The ammonium uptake and translocation and the K uptake rate of osamt1.1 mutants plants were also analyzed.A promoter of Pi-deficiency up-regulated gene OsPT6 and the promoter of a constitutive expression gene ubiquitin,with different expression pattern,were used for driving expression of ammonium transporter gene OsAMT1.1 in rice plants with the aim toincreasing ammonium uptake under Pi-deficiency conditions in the field,thus increasing P uptake and grain yield.We also analyzed the function of OsAMT2.1 in growth and development,and its involvement in ammonium uptake.The main results were as follows:1.Bioinformatic and qRT-PCR analysis showed that the open reading frame of OsAMT1.1 encods a protein composed of 498 amino acids.OsAMT1.1 is localized on plasma membrane and has 11 transmembranes(TMs).The expression of OsAMT1.1 in root and shoot showed a diurnal variation.Increasing ammonium and potassium supply could enhance the expression of OsAMT1.1 in root.2.Spatial expression pattern of OsAMT1.1 was detected in promoter-GUS transgenic rice in different tissues.GUS expression was constitutive in various tissues from roots to shoots.GUS-stained organs and their cross-sections showed strong expression of OsAMT1.1 in the primary roots,lateral roots,root-shoot junction,vascular bundles and leaf mesophyll cells,particularly in the root epidermis and stele parenchyma,leaf mesophyll and parenchyma cells of vascular bundles.3.Disruption of OsAMT1.1 gene expression decreased NH4+ uptake,root and shoot growth at both low-and high-NH4+ supply levels.OsAMT1.1 has a direct function in rice root NH4+ acquisition and contributes to about one quarter of net short-term NH4+ influx at both low and high 15NH4+ supply conditions.Knockout of OsAMT1.1 by CRISPR/Cas9 technology significantly decreased total N transport from root to shoot at low NH4+ level,it can be concluded that OsAMT1.1 is involved in the N translocation or osmat1.1 mutants showed more severed N deficiency at limited N supply condition.4.The osamt1.1 mutants in comparison to WT showed increase of potassium(K)absorption rate under high NH4+ condition but decrease under low NH4+ condition.The expression of both OsHAKl and OsHAK5 transporters,two known K transporters,were reduced under the NH4+-deficient supply and increased under the replete NH4+ in the osamt1.1 mutant roots compared with the WT.The change of expression patterns agreed with the alternation of root K absorption rate under the different NH4+ and K supplies.The mutants contained significant higher K in both roots and shoots at limited K(0.1 mM)supply but showed smaller phenotype with WT at 0.1 mM or ImM K supply when NH4+was replete,indicating that OsAMT1.1 contributes to the K and N homeostasis in rice.5.Ectopic expression of OsAMT1.1 gene driven by the promoter of OsPT6 encoding a low Pi-enhanced phosphate transporter led to increase OsAMT1.1 expression but still lower than the OsAMT1.1 expression driven by ubiquitin promoter(ubip:OsAMT1.1)under the Pi-deficiency.In comparison with WT and OsAMT1.1 overexpression transgenic plants,the OsPT6p:OsAMT1.1 transgenic lines showed increases of plant biomass and N uptake in the whole plants under low Pi supply condition.The field experiment with different Pi levels showed that OsPT6p:OsAMT1.1 plants grown better than WT in two Pi levels,the effective tiller numbers,total nitrogen and phosphate accumulation of whole plants were higher than WT and ubip:OsAMT1.1 transgenic plants.6.In root,the expression of OsAMT2.1 could be enhanced by supply of nitrogen and potassium.The expression of OsAMT2.1 in shoot showed a diurnal variation and up-regulated by K deficiency.The analyses of OsAMT2.1 T-DNA mutants showed that knockout of OsAMT2.1 did not significantly affect ammonium uptake by rice roots with low and normal ammonium supply,and has a slightly decrease compared with WT under high ammonium condition.This suggests that the OsAMT2.1 did not directly contribute to root ammonium uptake in rice.In long-term treatments,knockout mutation of OsAMT2.1 decreased the growth and nitrogen accumulation of whole plants at high NH4+ condition,but had no differences between WT at low NH4+ condition.In the field,knockout of OsAMT2.1 affect the plants net photosynthetic rate,stomatal conductance,and intercellular CO2 concentration and transpiration rate and resulted in decrease of effective numbers and accumulation of dry matter.Taken together,high-affinity ammonium transporter OsAMT1.1 contributes significantly to the NH4+ uptake from both low-and high-NH4+ environments and plays an important role in N-K homeostasis in rice.Enlarging ammonium uptake appropriately by expression of OsAMT1.1 driven by OsPT6p under low Pi conditions could be stimulating rhizosphere acidification and increasing N and P accumulation.The analyses of OsAMT2.1 mutants showed that knockout of OsAMT2.1 did not significantly affect ammonium uptake by rice roots with low-and sufficient-ammonium supply.However,knockout of OsAMT2.1 decreased the effective numbers and grain yield of rice might be related to the effect of plants photosynthesis and photorespiration.