Spacing Between NPA Domain Is a Key Regulatory Feature of Aquaporins for Silicon Transport

Silicon (Si) provides benefits to plants by enhancing resistance against biotic and abiotic stresses. The debate surrounding essentiality and benefits derived from Si is exacerbated by the differential ability of plant species to uptake the element from the soil. Uptake of Si is apparently higher in species carrying the specific aquaporins subclass nodulin 26-like intrinsic proteins (NIPs). In this study, we have characterized plant aquaporins to describe the features required for Si permeability. Thirty Si transporters were identified through comparative analysis of ca. 1000 aquaporins in 25 species with differing abilities to Si uptake. Our results showed that only species harbouring NIP-III aquaporins with a GSGR selectivity filter and a precise spacing of 108 amino acids (AA) between the NPA domains were able to absorb Si actively. The NPA spacing is of particular significance since it had never been considered in previous studies. Functional characterization performed using Xenopus oocyte expression system showed that only NIPs with 108 AA spacing exhibited Si permeability, while proteins differing in that distance did not. Furthermore, a Si transporter from poplar mutated into variants with 109- or 107-AA spacing failed to import, while a tomato NIP gene mutated from 109 to 108 AA exhibited a rare gain of function. The functionality of different tomato NIP and their mutant with varying spacing were also confirmed by heterologous expression in transgenic Arabidopsis plants. The study provides a basis for the understanding of NPA spacing evolution and its essentiality for Si permeability in plants.