Trafficking and intracellular localisation of Arabidopsis thaliana aquaporins

The majority of plant proteins essential for human and animal nutrition are synthesised and stored in the plant secretory pathway - a system of dynamic, membrane-bounded organelles comprising of the endoplasmic reticulum, Golgi complex, endosomes and vacuoles. In plants the protein storage vacuoles (PSV) are present in seeds whereas tissue lytic vacuoles (LV) are located in vegetative tissues. The vacuolar membrane, the tonoplast, contains aquaporin proteins that act as channels for the transport of water and small molecules; these are tonoplast intrinsic proteins (TIPs). The plasma membrane also contains aquaporins known as plasma membrane intrinsic proteins (PIPs). Research in our group has shown that these multi-pass transmembrane proteins are essential for the ability of seeds to germinate in conditions of water stress. To date, no definitive mechanism has been described by which TIP, and indeed most tonoplast proteins, are transported to the tonoplast. However, it is known how other aquaporins, including some PIPs, reach the plasma membrane. Using, as model proteins, a subset of aquaporins normally localised to either the tonoplast (TIP1;1) or the plasma membrane (PIP2;1), this project focussed on elucidating what determines the sorting of membrane proteins along the secretory pathway to the tonoplast. Following a systematic mutational approach of protein sequences of both TIP1;1 and PIP2;1 and subsequent imaging of fluorescent tagged proteins, although no singular distinct motif or domain of a TIP or PIP could be linked to localisation of the protein, the hypothesis that it is a combination of more than one factor remains. However, it was shown that a TIP with the N-terminal domain of a PIP still localises to the tonoplast thus implying the transmembrane domains of aquaporins are responsible, at least in part, to proper localisation of these proteins. Furthermore, the role of TIPs in regulating the germination of Arabidopsis seeds during conditions of drought and salt stress was investigated. By using Arabidopsis knockout lines, it was observed that the seed specific TIP3 isoforms are not only involved in water uptake but also in the control of seed germination. Finally, the root specific aquaporin TIP4;1, which has not previously been associated with germination, was found to play a role in regulation during drought stress