Discussion

No previous study has investigated the function of a rice member of the ALMT family in detail. Expression of all the OsALMTs was tested in the roots and leaves of rice plants and the OsALMT1 was chosen for further analysis because its expression was easily measured and because it sat on a separate branch of the phylogenetic tree (Figure 3.1). The expression of OsALMT2 could not detected in isolated RNA from leaves or roots and the sequence of OsALMT2 appeared to contain a truncation in the coding region which may render it non-functional. In contrast, OsALMT1 expression could easily be detected and thus a comprehensive description of OsALMT1 biology was possible. OsALMT1 was predicted to have all the attributes associated with the ALMT family including a PF11744 domain. Several algorithms predicted six transmembrane regions in the N-terminal half of the protein, but with various orientations of the N and C terminal ends towards either the inside or outside the cell. In general, ALMT members are predicted to have five to seven transmembrane regions in the N-terminal region and a long “tail” in the C-terminal half (see Chapter 1). Detailed topological analyses have been performed to further characterise the structure-function relationship of some ALMTs. The first experimental study of the secondary structure of TaALMT1 used an immunocytochemical method to analyse secondary structure. That study concluded that the C-terminal half (240 amino acids) is extracellular and the N-terminal region is also extracellular. There may also be an additional transmembrane domain in the N-terminus but this is unconfirmed (Motoda et al., 2007). More recent studies have questioned this topology and instead suggested that the C-terminal half of the TaALMT1 and other proteins is intracellular (Dreyer et al., 2012; Meyer et al., 2010; Mumm et al., 2013; Ryan et al., 2011). Similarly, experiments need to be undertaken to establish the secondary structure of OsALMT1 and to compare to the predicted secondary structures.

The on-line databases indicate that OsALMT1 is expressed in leaf, seedling and flower parts. Some other members of the ALMT family that are highly expressed in leaves are expressed in the guard cells and contribute to stomatal function. For example AtALMT6,

AtALMT9, AtALMT12 and HvALMT1 are highly expressed in the guard cells and the proteins encoded by these genes are important for guard cell function in Arabidopsis and barley (De Angeli et al., 2013b; Gruber et al., 2011; Gruber et al., 2010; Kovermann et al., 2007; Meyer et al., 2010; Meyer et al., 2011; Sasaki et al., 2010; Xu

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et al., 2015; Zhang et al., 2013; Zhang et al., 2014). All these genes are also expressed in other tissues. For instance, AtALMT6 is expressed in stem and flowers, AtALMT9 is expressed in the stem and along the root, AtALMT12 is expressed inseedlings and roots, while HvALMT1 is expressed in the root and in grain (Gruber et al., 2011; Kovermann

et al., 2007; Meyer et al., 2010; Meyer et al., 2011; Xu et al., 2015). The functions of all these proteins in tissues other than leaves are unclear except for HvALMT1 which facilitates malate efflux from the aleurone of grain during germination (Xu et al 2015).

The online databases explored here suggest that OsALMT1 expression in root tissue is affected by drought, heat and abscisic acid. Cis-elements involved in ABA and GA- responsive also can be found at the promoter region. Repeated copies of abscisic acid response element (ABRE) can confer ABA responsiveness to a minimal promoter, which proved to be involved in the regulation of various abiotic processes, including stomatal closure, seed and bud dormancy, and physiological responses to cold, drought, osmotic and salinity stresses (Kim et al., 2011; Narusaka et al., 2003). OsALMT1

expression was also predicted to change under different abiotic stresses such as drought, salt, cold, heat and P and Fe deficiency. The network predictions indicate the OsALMT1

expression is regulated by a transcription factor Os01g0625300 which is similar to heat shock transcription factor 31. Although the mechanism of stress activation of heat shock transcription factor 31 is not yet understood, the other heat shock transcription factor such as HSF1, 2, 3, 4 and 29 are proved to be linked to cellular signalling pathways under heat and oxidative stress conditions as well as functions not associated with stress such as cell cycle regulation, embryonic development, cellular differentiation, and spermatogenesis (Pirkkla et al., 2001; Zhong et al., 1998). These results indicated that the OsALMT1 might participant in plant responses to abiotic stresses involving hormones, light, heat, stress and defence responsiveness. Experiments should consider these treatments when exploring the possible function of the OsALMT1 protein in rice.

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CHAPTER 4