Role and control of HKT in Oryza sativa & Arabidopsis thaliana.

Salinity is a major abiotic stress influencing agricultural production in Australia and around the world. Plants grown in saline conditions are affected by both osmotic and ionic stress components. The focus of this thesis is on the ionic stress component of salinity stress and in particular the build up of sodium ions (Na⁺) in the leaf cytoplasm, one of the main components of salinity toxicity. In this thesis, genes encoding the high affinity potassium transporter family of proteins (HKTs) are studied in the plants Oryza sativa (rice) and Arabidopsis thaliana (Arabidopsis). These HKT transporters encode Na⁺ permeable membrane proteins and transport either Na⁺ selectively or co-transport Na⁺ and K⁺. HKT transporters have been identified in a number of plant species and to date have mainly been shown to be involved in reducing Na⁺ stress. A family of nine HKT genes have been identified in rice. Published reports to date have mainly investigated the function of these OsHKT genes in heterologous systems or have focused on one or two OsHKT genes in planta. During this PhD, tissue specific expression profiles were determined for each of the nine OsHKT genes, in ten rice varieties, exposed to a NaCl stress. Of most interest was OsHKT1;3 which showed very high levels of expression in leaf blades and sheath and higher levels of expression in NaCl treated roots compared to controls, across rice varieties. A range of experiments were designed to obtain further information regarding OsHKT1;3, as little was known about this gene or the encoded protein at the time of these experiments. A notable discovery was the identification of a novel OsHKT1;3 splice variant. In contrast to rice, Arabidopsis has only one HKT gene, AtHKT1;1. AtHKT1;1 is located in cells surrounding the xylem and is likely to be involved in the retrieval of Na⁺ from the xylem, thereby minimising shoot Na⁺ accumulation. To investigate the regulation of AtHKT1;1 gene expression two Arabidopsis ecotypes, Columbia-0 (Col-0) and C24, shown by QRT-PCR to have different AtHKT1;1 root expression levels, were studied. Sequencing of the C24 AtHKT1;1 promoter revealed substantial sequence differences between the Col-0 and C24 promoters, particularly 150 to 200 bp upstream of the AtHKT1;1 ATG start codon. It was hypothesised that these sequence differences were responsible for the lack of AtHKT1;1 root specific expression in C24 plants, due to a lack of transcription factor binding motif(s) or particular root specific transcription factor(s). To test this hypothesis a series of AtHKT1;1 promoter::GFP and AtHKT1;1 promoter::AtHKT1;1 cDNA constructs, with different combinations of Col-0 and C24 sequences, were tested. Preliminary results suggest that both the Col-0 and C24 AtHKT1;1 promoters are able to drive expression of the downstream transgene and therefore the sequence differences between the promoters is not the cause of the lack of C24 AtHKT1;1 root expression. A 1.6 kb insertion, identified in the second intron of the C24 AtHKT1;1 gene, is now proposed to disrupt C24 AtHKT1;1 root specific expression.Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 201