PHYSIOLOGICAL EFFECTS OF MG2+  DEFICIENCY AND AL3+ TOXICITY UPON ARABIDOPSIS PLANTS

Get Complete Project Material File(s) Now! »

Bacterial Mg2+ transport

CorA

CorA is the best characterized of the Mg2+ transport genes and encodes the main Mg2+ uptake system in Salmonella typhimurium, Escherichia coli and Methanococcus jannaschii (Hmiel et al., 1986; Smith et al., 1998a). A number of genes from different organisms have weak homology to CorA (see Table 1.1) and all contain a GMN motif within their predicted transmembrane domains (Kehres et al., 1998). CorA transports Mg2+ against its concentration gradient and CorA is sensitive to the membrane potential, with hyperpolarization activating its activity (Kehres and Maguire, 2002; Froschauer et al., 2004). CorA is constitutively expressed and is not regulated by external Mg2+, although the structure of the CorA protein (described in Figure 1.2) suggests that it may be regulated by intracellular Mg2+ (Snavely et al., 1991; Chamnongpol and Groisman, 2002; Moomaw and Maguire, 2008). Snavely et al. (1989) found that CorA is also essential for Mg2+ efflux in S. typhimurium, and Mg2+ efflux has also been observed with expression of the homologous yeast genes ALR1 and MRS2 (Liu et al., 2002; Kolisek et al., 2003). Furthermore, the CorA-type protein ZntB (Worlock and Smith, 2002) has recently been shown to have very similar structure to CorA in its cytosolic domain (Tan et al., 2009) and is primarily responsible for Zn2+ efflux in S. typhimurium,
suggesting that efflux may be an important feature of at least some CorA-type transporters.
Besides Mg2+, CorA has also been shown to transport Co2+ and Ni2+ with affinities of 30 μM and 240 μM respectively, although in S. typhimurium and E. coli these ions are considered to be physiologically irrelevant (Hmiel et al., 1986; Gibson et al., 1991; Snavely et al., 1991). Phylogenetic analyses have shown that CorA is widely dispersed in Eubacteria and many species of Archea (Smith and Maguire, 1995; Kehres et al., 1998; Froschauer et al., 2004). In 2006, three separate groups published the crystal structure of CorA from the thermophillic The relationship 6 between magnesium status and aluminium toxicity in Arabidopsis bacterium Thermotiga maratima, with 1.85-3.9 Å resolution (described in Figure 1.2, Eshaghi et al., 2006; Lunin et al., 2006; Payandeh and Pai, 2006). Two of these groups described a hydrated cation, consistent with Mg2+, bound to the intracellular domain of CorA (Lunin et al., 2006; Payandeh and Pai, 2006), while the third group described binding of both Co2+ and Mg2+ intracellularly (Eshaghi et al., 2006). Interestingly, recent attempts by Eschagi and co-workers to generate mutants with altered Mg2+ kinetics based on this structure have shown that in T. maratima CorA is intracellularly regulated by Co2+ and transports Co2+ with 100-fold greater affinity than Mg2+ (Xia et al., 2011). This shows that CorA, while conserved across bacteria, has differential selectivity according to species and extremophillic bacteria like T. maritima may have differences in their CorA sequence and structure due to a physiological preference for Co2+ instead of Mg2+ (Niegowski and Eshaghi, 2007; Xia et al., 2011).

MgtA and MgtB

Three other Mg2+ transport proteins are known to exist in bacteria, which are unrelated to CorA. Although they transport Mg2+ down its electrochemical gradient, MgtA and MgtB are P-type ATPases and MgtA and MgtB gene expression is up-regulated in response to low levels of extracellular Mg2+, via the PhoPQ system (Snavely et al., 1989; Snavely et al., 1991; Smith et al., 1998b; Groisman, 2001; Chamnongpol and Groisman, 2002). PhoQ senses low extracellular Mg2+, phosphorylating PhoP, which in turn promotes MgtA/B gene expression (Groisman, 2001). MgtA also contains a stem-loop structure in its RNA 5’-untranslated region (5’-UTR) which has been shown to act as a riboswitch which senses intracellular concentrations of Mg2+ (Cromie et al., 2006). Both MgtA and MgtB are up-regulated during infection with Salmonella typhimurium and although they are not required for pathogenicity, they improve pathogen survival, perhaps by compensating for the reduced Mg2+-uptake capacity of CorA under very low Mg2+ conditions (Moncrief and Maguire, 1998; Smith et al., 1998b). MgtA and MgtB have 50% identity and have slight differences in selectivity for Mg2+ and inhibition by other cations (Snavely et al., 1989). Both proteins are found in a variety of bacterial species, with MgtA more common than MgtB (Blanc-Potard and Groisman, 1997).

MgtE

Unrelated to the other bacterial Mg2+ transport genes, MgtE was identified from genomic libraries of Providencia stuartii and Bacillus firmus by complementation of the S. typhimurium corA mgtA mgtB mutant MM281 (Smith et al., 1995; Townsend et al., 1995). The primary function of MgtE protein is unclear, although it has functional similarities to both CorA and MgtA/B (Kehres and Maguire, 2002). Like CorA, MgtE is constitutively expressed and mediates Mg2+ and Co2+ influx (Snavely et al., 1989). The crystal structure of MgtE revealed that it has five transmembrane domains and exists as a homodimer (Hattori et al., The relationship 8 between magnesium status and aluminium toxicity in Arabidopsis 2007). Although CorA and MgtE are structurally distinct they also share several similarities; each has a total of ten TM domains forming a membrane pore, a large cytosolic domain and alpha helices which are thought to shift orientation upon the binding of intracellular Mg2+ (reviewed in Moomaw and Maguire, 2008). Therefore it is thought that intracellular Mg2+ regulates the MgtE channel in a similar way to that expected for CorA. However unlike CorA, intracellular Mg2+ also regulates MgtE RNA expression via a metal-induced RNA folding domain on its 5’-UTR similar to that observed for MgtA (Cromie et al., 2006; Dann et al., 2007).
MgtE-domain containing proteins are found in Eubacteria and Archea, although they are not as widely distributed as CorA. They have also been identified in Metazoa, in Caenorhabditis elegans, Drosophila melanogaster, mouse and humans (Wabakken et al., 2003). The human MgtE gene, SLC41, is expressed in all tissue types, in particular the heart and testis (Wabakken et al., 2003). Quamme and co-workers have shown that SLC41 mediates Mg2+ transport at physiological Mg2+ concentrations and its expression is up-regulated in response to low Mg2+ (Goytain and Quamme, 2005b, c).

Yeast Mg2+ transport

Alr proteins

ALR1 and ALR2 were identified from a screen for genes conferring increased Al3+ tolerance when overexpressed in Saccharomyces cerevisiae (MacDiarmid and Gardner, 1998). Alr1 is localized to the plasma membrane and is required for yeast growth under normal (4 mM Mg2+) conditions and allows yeast growth on Mg2+ concentrations as low as 20 μM (Lee, 2006). Besides Mg2+, Alr1 also transports Co2+, Ni2+, Mn2+ and Zn2+ and exhibits sensitivity to Ca2+, Cu2+ Al3+ and La2+ (MacDiarmid and Gardner, 1998). Electrophysiological work using patch-clamp of yeast protoplasts expressing ALR1 detected dual Mg2+ currents, suggesting that Alr1 may also mediate Mg2+ efflux (Liu et al., 2002). Yeast overexpressing ALR2 have similar growth to yeast with native ALR1 expression levels, which led to the suggestion that Alr2 may be a functionally redundant copy of Alr1 (MacDiarmid, 1997). However, more recent work by the same author suggests that Alr2 may modulate Alr1 activity depending on the yeast strain used (Pisat et al., 2009). This may occur through protein interactions; Schweyen and co-workers found that Alr2 has a dominant negative effect on Alr1 activity, which is associated with the formation of hetero-oligomers containing both proteins (Wachek et al., 2006).

CHAPTER ONE | INTRODUCTION 
1.1 Magnesium in biological systems
1.2 Mg2+ uptake and distribution in plants
1.3 Molecular mechanisms of Mg2+ uptake and transport
1.4 Mg2+ deficiency
1.5 Mg2+ toxicity
1.6 Aluminium3+ and biological systems
1.7 Physiological effects of Al3+ on plants
1.8 Molecular features of Al3+ toxicity
1.9 Mg2+ in Al3+ toxicity
1.10 Al3+ tolerance in plants
1.11 Aims of this work
CHAPTER TWO | MATERIALS AND METHODS 
2.1 General reagents
2.2 Bacterial growth and manipulation
2.3 Yeast growth and manipulation
2.4 Plant Growth and manipulation
2.5 DNA analysis and manipulation
2.6 RNA analysis
2.7 Protein analysis
2.8 Microscopy
2.9 Flame atomic absorption spectroscopy (Flame AAS)
CHAPTER THREE | PHYSIOLOGICAL EFFECTS OF MG2+  DEFICIENCY AND AL3+ TOXICITY UPON ARABIDOPSIS PLANTS
3.1 Introduction
3.2 Mg2+ starvation of Arabidopsis plants grown in hydroponic culture
3.3 Mg2+ starvation in submerged culture in Schott bottles
3.4 Mg2+ uptake by Mg2+-starved plants
3.5 Recovery of Arabidopsis plants following Mg2+ starvation
3.6 Responses of submerged plants to Al3+
3.7 Al3+ toxicity in non-submerged conditions
3.8 Mg2+ uptake in the presence of Al3+
3.9 Effects of Al3+ on root morphology
3.10 Plant Mg2+ status affects external media pH
3.11 Discussion
CHAPTER FOUR | EXPRESSION OF MICROBIAL MG2+  TRANSPORT GENES ALR1 AND CORA IN ARABIDOPSIS
4.1 Introduction
4.2 A series of ALR1- and CorA-ased constructs were generated in the yeast plasmid pYES3
4.3 Function of the Mg2+ transport genes was checked by expression in yeast
4.4 Generation of Agrobacterium binary vector constructs for expression in plants
4.5 Arabidopsis thaliana was transformed with each of the plant expression constructs and transgenic lines selected
4.6 Expression of 35S::CorA reduces plant health and survival
4.7 The reconstruction of ALR1 was effective in removing the majority of cryptic plant post-transcriptional processing
4.8 CorA and crALR1 fusion gene expression and protein detection
4.9 Discussion
CHAPTER FIVE | PHENOTYPIC RESPONSES OF  CORA AND CRALR1 TRANSGENIC PLANTS TO MG2+
5.1 Introduction
5.2 CorA plants have a Mg2+-deficiency phenotype
5.3 Homozygous CorA plants have elevated Mg2+
5.4 Mg2+-starved ‘CorA’ plants have increased Mg2+ uptake rates
5.5 Phenotypes of CorA and crALR1 plants grown in limited Mg2+
5.6 CorA plants have improved tolerance to toxic levels of Mg2+
5.7 Effects of other divalent cations on transgenic plant lines
5.8 Discussion
CHAPTER SIX | PHENOTYPIC RESPONSES OF 205 CORA AND CRALR1 TRANSGENIC PLANTS TO Al3+
CHAPTER SEVEN | EFFECTS OF MG2+ AND AL3+ ON THE 233 ARABIDOPSIS TRANSCRIPTOME
CHAPTER EIGHT | CONCLUDING DISCUSSION 
REFERENCES

GET THE COMPLETE PROJECT
THE RELATIONSHIP BETWEEN MAGNESIUM STATUS AND ALUMINIUM TOXICITY IN ARABIDOPSIS