Cloning and characterisation of genes encoding phosphate and sulphate transporters from rice

Godwin, Rosamond Mary. (2002). Cloning and characterisation of genes encoding phosphate and sulphate transporters from rice PhD Thesis, School of Land, Crop and Food Sciences, The University of Queensland.

       
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Author Godwin, Rosamond Mary.
Thesis Title Cloning and characterisation of genes encoding phosphate and sulphate transporters from rice
School, Centre or Institute School of Land, Crop and Food Sciences
Institution The University of Queensland
Publication date 2002
Thesis type PhD Thesis
Supervisor Frank Smith
Bemie Carroll
Total pages 147
Collection year 2002
Language eng
Subjects L
300203 Plant Improvement (Selection, Breeding and Genetic Engineering)
620103 Rice
Formatted abstract As population pressures and environmental degradation increase, it is important to make agricultural systems more sustainable. Phosphorus and sulphur are significant macronutrients which are often deficient and/or poorly available in many agricultural soils and therefore can limit the yield and quality of agricultural products. Enhancing the efficiency with which plants acquire and use available nutrients is an important component in making our agricultural systems more sustainable, reducing the need for costly applications of fertilisers, and decreasing environmental damage through runoff. To improve the efficiency of nutrient use in plants it is necessary to understand how uptake and subsequent distribution of nutrients among plant parts is mediated and how these processes are regulated. Considerable advances have been made in the last 10 years in our understanding of the phosphate and sulphate transport in plants. This has resulted from the application of molecular biology to plant nutrition. Rice, (Oryza sativa) is one of the world's most important food crops being the primary food source for at least one third of the world's population.  

In the past there has been little research into the molecular mechanisms of nutrient uptake and regulation in rice. The aim of this project was to clone and characterise genes from rice that are involved in phosphate and sulphate transport in order to enhance our understanding of the molecular nature of nutrient transport processes. Genes were isolated from a rice genomic library and their cDNAs generated using reverse-transcription polymerase chain reaction (RT-PCR).The genes were then characterised in terms of their expression in tissues and in response to different nutrient status. Functional analyses of the cloned transporter genes was also attempted using expression in yeast to study kinetic characteristics.

Four rice genes were isolated and cloned, two encoding phosphate transporters and two encoding sulphate transporters. Southern analyses revealed that each of these genes occur in single copy in the rice genome. OsPT1 is predicted to encode a protein of 555 amino acids with a molecular mass of 6lkD while OsPT2 is predicted to encode a protein of 538 amino acids with a molecular mass of 60kD. Both genes encode putative phosphate transporters belonging to the Phtl family of plant H+/ H2P04- symporters. They possess between 72-87% similarity and 52-79% identity with other plant homologues, have conserved signature sequences for the Pht1 family oftransporters, and have a predicted topology of 12 membrane spanning domains of α helices. OsPT2 was found to be expressed in roots and shoots with expression being unresponsive to external phosphorus supply. Partial complementation of a yeast strain, deficient in high affmity phosphate uptake, was achieved with the product encoded by OsPT2, thereby demonstrating function of the gene. This was detected using a sensitive test for acid phosphatase activity. Under conditions tested in this project, northern analyses did not detect expression of OsPT1, however sequence analyses suggest that it is also a functioning gene. Further experimentation would be needed to determine expression patterns and role of OsPT1

OsST1and OsST2 encode putative sulphate transporters. OsST1 is predicted to encode a transporter of 662 amino acids and molecular mass of 74kD while OsST2 is predicted to encode a transporter of 689 amino acids and a molecular mass of 77kD. Both genes encode proteins with predicted topologies of 12 membrane spanning domains and possess signature sequences of the H+/ H2S042- symporter family of transporters. The two rice sulphate transporter genes exhibit between 61-90% similarity and 36-69% identity with other plant homologues. OsST1 is strongly expressed in roots with levels of expression being strongly enhanced by sulphate starvation. In situ hybridisation experiments reveal that expression of OsST1 is localised in the main absorptive region of roots especially in all cells within a few millimetres of the root tip, and the tips of emerging laterals. It is likely that the transporter encoded by OsST1 is responsible for uptake of sulphate from the soil solution.

OsST2 is predicted to be a plastidic sulphate transporter possessing a typical plastid-targeting transit peptide. Expression of OsST2 in both roots and shoots was unresponsive to the sulphur status of the plant. The presumed function of OsST2 in shoots is to transport sulphate across plastid membranes to active sites of sulphate reduction. Functional analyses of the cloned nee transporters was not successful in a heterologous yeast expression system. This is probably because the rice transporters were not recognised by the yeast or correctly targeted to the cell membrane. It is also possible that other proteins interact with transporters in each species to provide a functional unit and these may not be present in the yeast. It is important therefore to develop other expression systems in order to study functional characteristics of plant transporters.

Information gained in this project provides a sound basis for further studies involving phosphate and sulphate transport in rice. With further experimentation it will be possible to clone additional genes encoding phosphate and sulphate transporters and conduct detailed analyses of their localisation and regulation. Elucidation of the mechanisms controlling uptake and transport of these nutrients in rice can therefore be achieved and this knowledge extended to other cereal crops. This will aid the genetic manipulation of transport systems in order to improve efficiency of nutrient use in plants. 
Keyword Rice -- Genetics
Cloning

 
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Created: Fri, 24 Aug 2007, 18:01:49 EST