Plant aquaporins that facilitate cation transport and their regulation by phosphorylation
Date
2020
Authors
McGaughey, Samantha
Editors
Advisors
Byrt, Caitlin
Tyerman, Steve
Tyerman, Steve
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Thesis
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Abstract
Plants respond to osmotic stresses by adjusting the regulation of water and ion transport mechanisms. A subset of aquaporins are candidates for being involved in these mechanisms. For example, two Arabidopsis Plasma membrane Intrinsic Proteins (PIPs), AtPIP2;1 and AtPIP2;2, were previously reported to be water and ion transporting aquaporins. Here, the ion and water transport properties of sets of aquaporins from different plant species were investigated using the heterologous expression systems Xenopus laevis oocytes and yeast (Saccharomyces cerevisiae). These sets included PIP isoforms from the model dicot Arabidopsis, the model monocot Setaria (Setaria viridis) and the agricultural crop barley (Hordeum vulgare); and a candidate Tonoplast Intrinsic Protein (TIP) aquaporin from barley, HvTIP2;2. One or more aquaporins from each different plant species was found to facilitate ion transport in heterologous systems. The identification of ion transporting aquaporins in a range of plant species, including dicot and monocot species, indicates that this feature could have had an early evolutionary origin that preceded monocot-dicot divergence 140-170 million years ago. To further test the hypothesis of an early evolutionary origin for ion transporting aquaporins a PIP-like aquaporin from a filamentous terrestrial alga was tested and confirmed to facilitate water and ion transport. AtPIP2;1-associated permeability properties are similar to previously reported features associated with non-selective cation channels (NSCCs), and AtPIP2;1 has been proposed as an NSCC molecular candidate. Testing of AtPIP2;1 permeability revealed that it can facilitate the transport of a range of monovalent cations, such as sodium (Na+), potassium (K+), cesium (Cs+) and rubidium (Rb+), and AtPIP2;1 permeability is influenced by changes in calcium, pH and treatments involving addition of cyclic nucleotides (cNMPs). Ion transporting PIP2;1 homologs in cereals may also be candidates for NSCCs. The PIPs from Setaria were tested for boric acid and hydrogen peroxide (H2O2) transport and it was observed that in general the transport of H2O2 and ions occurred for different sets of PIP isoforms. The influence of post-translational modifications on AtPIP2;1 ion transport was investigated using site directed mutagenesis to mimic different phosphorylation states. Previously, the phosphorylation status of several serine (S) residues on the C-terminal domain (S280 and S283) and the intracellular loop B (S121) and loop D (S194) of AtPIP2;1, were linked to salt stress responses and monomeric pore gating, respectively. The phosphorylation state of these sites regulated AtPIP2;1 water and ion channel function when expressed in heterologous systems and influenced whether AtPIP2;1 functioned primarily as a water channel or an ion channel. Candidate upstream kinases OST1 (Snrk2.6), and CDPKs CPK3 and CPK21 were investigated for their capacity to influence AtPIP2;1-associated water and ion transport in X. laevis oocytes. Co-expression with OST1 and CPK3 reduced both the water and ion transport of AtPIP2;1, whereas CPK21 co-expression only influenced AtPIP2;1 water channel activity. Ion transporting plant aquaporins are candidates for NSCC and ion and water co-transport mechanisms, and they may contribute to osmotic stress tolerance mechanisms. The potential physiological roles of plant ion transporting aquaporins and options for future experiments are discussed.
School/Discipline
School of Agriculture, Food and Wine
Dissertation Note
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2020
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