Characterising Tobacco Aquaporins: towards engineering improved photosynthesis and plant performance

Abstract

Future climatic conditions and an increasing global world population require the development of higher yielding and more resilient crops. Aquaporins are increasingly being studied as genetic engineering targets to tackle food security challenges. They constitute a major family of membrane spanning channel proteins, selectively facilitating the passive bidirectional passage of a range of solutes essential for numerous plant processes, including water relations, growth and development, stress responses, root nutrient uptake, and photosynthesis. In plants, aquaporins occur in five major subfamilies that differ in temporal and spatial gene expression, subcellular protein localisation, substrate specificity, and post-translational regulatory mechanisms, collectively providing a dynamic transportation network spanning the entire plant. Of particular interest are aquaporins in the Plasma membrane Intrinsic Proteins (PIP) subfamily, some of which have been shown to enhance membrane permeability to CO2. This role could influence photosynthetic efficiency, which is limited by the ease with which CO2 diffuses across cellular membranes from intercellular airspaces to the site of fixation within the chloroplast (i.e. mesophyll conductance). As such, PIP aquaporins are an attractive target for engineering enhanced photosynthesis. The ability to manipulate aquaporins towards improving plant productivity is reliant on expanding our insight into their diversity and functional roles.

My PhD project contributes towards a better understanding of aquaporin biology, providing a comprehensive overview of the Nicotiana tabacum (tobacco) aquaporin family. Tobacco is a popular model system capable of scaling from the laboratory to the field, it is closely related to several major economic crops (e.g. tomato, potato, eggplant and capsicum) and itself has new commercial applications. To date one PIP gene has been described in tobacco to be a CO2 pore, NtAQP1. However, beyond this there is little characterization of tobacco aquaporins in the literature.

We identified that the tobacco genome encodes 76 aquaporins, making it the second largest characterised aquaporin family after that of Brassica napus, containing 121 genes. Tobacco aquaporins fall into five distinct subfamilies, for which we characterised phylogenetic relationships, gene structures, protein sequences, selectivity filter compositions, sub-cellular localisation, and tissue-specific expression. Once the tobacco aquaporin family was established, we functionally characterized a diverse subset of candidate aquaporins, determining their sub-cellar localisation in planta and their substrate specificities through yeast-based functional assays developed within our laboratory. These novel yeast-based assays allowed us to test aquaporin permeability for a range of substrates important for plant function (water, hydrogen peroxide, urea and boron). 3D protein homology modelling was then used for an integrated aquaporin characterisation, linking substrate specificities and amino acid primary sequence to the pores' radius and physico-chemical properties.

Towards enhancing photosynthesis, we identified several PIP aquaporins that are likely candidates for transporting CO2 and constitutively over-expressed these in tobacco. Gas exchange measurements showed gene-specific alterations to photosynthesis. Although enhanced photosynthetic rates were observed with PIP1 over expression, no significant increase in mesophyll conductance was observed. Further investigations are needed to re-examine the consequence of overexpression of NtAQP1 on mesophyll conductance and clarify the possible pleiotropic effects that constitutive over-expression of tobacco PIPs has on leaf properties and plant growth.