Exploring functional diversity of Nicotiana tabacum Aquaporins

Aquaporins (AQPs) are multifunctional membrane proteins which have greatly diversified in number and function in the plant Kingdom. In plants, AQPs have evolved to comprise a dynamic solute transport network occurring in all tissues and facilitating transport of water and vital solutes across various cellular membranes. Plant AQPs are involved in a multitude of plant physiological processes, however a better understanding is required of AQP structure-function relationships, multifunctionality and cell membrane localisation in order to begin to describe putative functional roles for the numerous plant AQP gene isoforms. Using an integrated approach, we characterised nine diverse Nicotiana tabacum (tobacco) aquaporins, spanning the 3 largest AQP subfamilies (PIP, TIP, and NIP) and with varied gene expression profiles. High-throughput yeast-based functional screens identified novel candidates for water, hydrogen peroxide (H2O2), boric acid (BA) and urea transport across the 3 AQP subfamilies. Using GFP translational fusions, AQPs observed in planta were localised to the plasma membrane, tonoplast and endoplasmic reticulum. AlphaFold protein models illustrated differences in pore shape and size across subfamilies. Our analysis supports the importance of functional data for deciphering unknown AQP structure-function relationships and uncovering novel candidates for in planta solute transport.