Molecular physiology of ammonium transport in rice

Abstract

The availability of nitrogen in the soil is one of the main limiting factors for plant productivity, biomass accumulation and crop yield. Well-aerated soils are relatively rich in N03- and poor in NH4 +, whereas in anaerobic soils of irrigated or rain-fed lowland rice, NH4 + is the most prevalent source of nitrogen. Ammonium uptake is presumed to be controlled by at least two gene families in plants, namely AMTJ and AMT2. Each gene family consists of one to several members, some of which have been cloned in Arabidopsis, tomato and rice. As in Arabidopsis, rice is predicted to have multiple members for the AMTJ gene family. Preliminary studies of an ammonium transporter (OsAMTl;l) in rice (cv. Taipei 309), presented in this thesis, revealed its expression in roots and mature leaves under different nitrogen regimes. Expression levels were generally higher in roots than in leaves. The project was aimed at providing an insight into rice AMT ( OsAMT) genes, their regulation and their physiological roles in rice. A transgenic approach was undertaken to investigate the role of OsAMTl m ammonium uptake and consequent ammonium assimilation under different nitrogen regimes. Two expression cassettes were made using the full-length eDNA of OsAMTl;l to have their sense or antisense expression driven by the maize ubiquitin promoter. Transgenic lines were produced from two rice cultivars, namely Taipei 309 and Jarrah (an Australian cultivar), by Agrobacterium-mediated transformation using these expression cassettes mounted on the binary vector pWBVec8 that contained a hygromycin resistance gene (hph) as the selectable marker. Only a small number of Taipei 309 transgenic lines could be regenerated with the antisense transgene, most of which were sterile and none showed any down-regulation of the endogenous OsAMTl;l mRNA. No antisense transgenic lines could be regenerated from cv. Jarrah The Ubil(l) promoter-driven OsAMTl;l sense transgene in cvs. Taipei 309 and Jarrah increased OsAMTl;l transcript levels, which positively correlated with transgene copy number. Under both ammonium-fed and ammonium-starved growth condtions, ammonium-induced depolarisation of root cell plasmamembrane electrical potentials (Em) were substantially greater in OsAMTl; 1 over-expressing transgenic lines compared to wild type plants. These transgenic plants showed increased ammonium uptake and root ammonium content, however, they had decreased biomass especially when grown under high concentrations of NH4 +. A low stringency Southern blot hybridization of genomic DNA isolated from wild type Taipei 309 with radioactively-labelled full length OsAMTl;l eDNA showed 8-10 hybridizing bands. Using a similar low stringency hybridization approach, genomic clones of three members of the OsAMTl family, including the OsAMTl;l, were isolated. Sequence data from these clones revealed that all three were intronless, at least in the coding region. These represent the first genomic clones of ammonium transporters isolated from rice. The DNA sequences of the predicted coding regions of these genes showed 98.6%, 81% and 73% homology to that of OsAMTl; 1 from cv Nipponbare. On the basis of the presence of several promoter signals, such as TAT A and CAAT boxes, a 2.4 kb sequence upstream of the ATG codon from OsAMTl;l, a 2.5 kb fragment from clone OsAMT1;2, and a 1.4 kb fragment from clone OsAMT1;3, were selected as putative promoter regions and cloned in front of the reporter gene UidA. These constructs were then used to transform rice cv. Taipei 309 to study the expression and regulation of these ammonium transporter genes. Determination of the expression pattern of GUS in resulting transgenic lines would allow the location and level of expression of the different OsAMT genes under various developmental and environmental conditions to be measured. Due to time constrains, results from these experiments could not be included in this thesis.