MtNIP5;1, a novel Medicago truncatula boron diffusion facilitator induced under deficiency

Background: Boron (B) deficiency or toxicity in soils can cause the inhibition of growth and an altered development in plants. In such conditions, the participation of two distinct protein families (the aquaporin family Major intrinsic proteins (MIPs) and the Boron transporter family BOR) is required to minimize detrimental effects caused by B stress. Legumes comprise important crops that offer major agronomic benefits, including the capacity of establishing symbiosis with rhizobia fixing atmospheric N2 and for being important food protein sources. It has been proven that legumes are susceptible to B stress, which leads to important yield penalties. However, little is known about the transport mechanisms responsible for B uptake and distribution in legumes, especially under deficiency.Results: A Medicago truncatula protein, MtNIP5;1 (Medtr1g097840) (homologous to the Arabidopsis thaliana AtNIP5;1) was identified as a novel legume B transporter involved in B uptake under deficiency. Further analyses revealed that this M. truncatula aquaporin expression was boron-regulated in roots, being induced under deficiency and repressed under toxicity. It localizes at the plasma membrane of root epidermal cells and in nodules, where B plays pivotal roles in symbiosis. Furthermore, a partial complementation of the nip5;1 A. thaliana mutant phenotype under B deficiency supports a functional role of MtNIP5;1 as a B transporter in this legume model plant. Conclusions: The results here presented support a functional role of MtNIP5;1 in B uptake under deficiency and provides new insights into this micronutrient transport mechanisms in legume species.

Insights into the Selectivity Mechanisms of Grapevine NIP Aquaporins

Nodulin 26-like intrinsic proteins (NIPs) of the plant aquaporin family majorly facilitate the transport of physiologically relevant solutes. The present study intended to investigate how substrate selectivity in grapevine NIPs is influenced by the aromatic/arginine (ar/R) selectivity filter within the pore and the possible underlying mechanisms. A mutational approach was used to interchange the ar/R residues between grapevine NIPs (VvTnNIP1;1 with VvTnNIP6;1, and VvTnNIP2;1 with VvTnNIP5;1). Their functional characterization by stopped-flow spectroscopy in Saccharomyces cerevisiae revealed that mutations in residues of H2/H5 helices in VvTnNIP1;1 and VvTnNIP6;1 caused a general decline in membrane glycerol permeability but did not impart the expected substrate conductivity in the mutants. This result suggests that ar/R filter substitution could alter the NIP channel activity, but it was not sufficient to interchange their substrate preferences. Further, homology modeling analyses evidenced that variations in the pore radius combined with the differences in the channel’s physicochemical properties (hydrophilicity/hydrophobicity) may drive substrate selectivity. Furthermore, yeast growth assays showed that H5 residue substitution alleviated the sensitivity of VvTnNIP2;1 and VvTnNIP5;1 to As, B, and Se, implying importance of H5 sequence for substrate selection. These results contribute to the knowledge of the overall determinants of substrate selectivity in NIPs.

Covalently Linking Oligomerization-Impaired GlpF Protomers Does Not Completely Re-establish Wild-Type Channel Activity

Integral membrane proteins of the aquaporin family facilitate rapid water flux across cellular membranes in all domains of life. Although the water-conducting pore is clearly defined in an aquaporin monomer, all aquaporins assemble into stable tetramers. In order to investigate the role of protomer–protomer interactions, we analyzed the activity of heterotetramers containing increasing fractions of mutated monomers, which have an impaired oligomerization propensity and activity. In order to enforce interaction between the protomers, we designed and analyzed a genetically fused homotetramer of GlpF, the aquaglyceroporin of the bacterium Escherichia coli (E. coli). However, increasing fractions of the oligomerization-impaired mutant GlpF E43A affected the activity of the GlpF heterotetramer in a nearly linear manner, indicating that the reduced protein activity, caused by the introduced mutations, cannot be fully compensated by simply covalently linking the monomers. Taken together, the results underline the importance of exactly positioned monomer–monomer contacts in an assembled GlpF tetramer.