Functional evolution of nodulin 26-like intrinsic proteins: from bacterial arsenic detoxification to plant nutrient transport.

Nodulin 26-like intrinsic proteins (NIPs) play essential roles in transporting the nutrients silicon and boron in seed plants, but the evolutionary origin of this transport function and the co-permeability to toxic arsenic remains enigmatic. Horizontal gene transfer of a yet uncharacterised bacterial AqpN-aquaporin group was the starting-point for plant NIP evolution. We combined intense sequence, phylogenetic and genetic context analyses and a mutational approach with various transport assays in oocytes and plants to resolve the transorganismal and functional evolution of bacterial and algal and terrestrial plant NIPs and to reveal their molecular transport specificity features. We discovered that aqpN genes are prevalently located in arsenic resistance operons of various prokaryotic phyla. We provided genetic and functional evidence that these proteins contribute to the arsenic detoxification machinery. We identified NIPs with the ancestral bacterial AqpN selectivity filter composition in algae, liverworts, moss, hornworts and ferns and demonstrated that these archetype plant NIPs and their prokaryotic progenitors are almost impermeable to water and silicon but transport arsenic and boron. With a mutational approach, we demonstrated that during evolution, ancestral NIP selectivity shifted to allow subfunctionalisations. Together, our data provided evidence that evolution converted bacterial arsenic efflux channels into essential seed plant nutrient transporters.

Heterotetramerization of Plant PIP1 and PIP2 Aquaporins Is an Evolutionary Ancient Feature to Guide PIP1 Plasma Membrane Localization and Function.

Aquaporins (AQPs) are tetrameric channel proteins regulating the transmembrane flux of small uncharged solutes and in particular water in living organisms. In plants, members of the plasma membrane intrinsic protein (PIP) AQP subfamily are important for the maintenance of the plant water status through the control of cell and tissue hydraulics. The PIP subfamily is subdivided into two groups: PIP1 and PIP2 that exhibit different water-channel activities when expressed in Xenopus oocytes or yeast cells. Most PIP1 and PIP2 isoforms physically interact and assemble in heterotetramers to modulate their subcellular localization and channel activity when they are co-expressed in oocytes, yeasts, and plants. Whether the interaction between different PIPs is stochastic or controlled by cell regulatory processes is still unknown. Here, we analyzed the water transport activity and the subcellular localization behavior of the complete PIP subfamily (SmPIP1;1, SmPIP2;1, and SmPIP2;2) of the lycophyte Selaginella moellendorffii upon (co-)expression in yeast and Xenopus oocytes. As observed for most of the PIP1 and PIP2 isoforms in other species, SmPIP1;1 was retained in the ER while SmPIP2;1 was found in the plasma membrane but, upon co-expression, both isoforms were found in the plasma membrane, leading to a synergistic effect on the water membrane permeability. SmPIP2;2 behaves as a PIP1, being retained in the endoplasmic reticulum when expressed alone in oocytes or in yeasts. Interestingly, in contrast to the oocyte system, in yeasts no synergistic effect on the membrane permeability was observed upon SmPIP1;1/SmPIP2;1 co-expression. We also demonstrated that SmPIP2;1 is permeable to water and the signaling molecule hydrogen peroxide. Moreover, growth- and complementation assays in the yeast system showed that heteromerization in all possible SmPIP combinations did not modify the substrate specificity of the channels. These results suggest that the characteristics known for angiosperm PIP1 and PIP2 isoforms in terms of their water transport activity, trafficking, and interaction emerged already as early as in non-seed vascular plants. The existence and conservation of these characteristics may argue for the fact that PIP2s are indeed involved in the delivery of PIP1s to the plasma membrane and that the formation of functional heterotetramers is of biological relevance.

Genome-wide identification of aquaporin encoding genes in Brassica oleracea and their phylogenetic sequence comparison to Brassica crops and Arabidopsis.

Aquaporins (AQPs) are essential channel proteins that regulate plant water homeostasis and the uptake and distribution of uncharged solutes such as metalloids, urea, ammonia, and carbon dioxide. Despite their importance as crop plants, little is known about AQP gene and protein function in cabbage (Brassica oleracea) and other Brassica species. The recent releases of the genome sequences of B. oleracea and Brassica rapa allow comparative genomic studies in these species to investigate the evolution and features of Brassica genes and proteins. In this study, we identified all AQP genes in B. oleracea by a genome-wide survey. In total, 67 genes of four plant AQP subfamilies were identified. Their full-length gene sequences and locations on chromosomes and scaffolds were manually curated. The identification of six additional full-length AQP sequences in the B. rapa genome added to the recently published AQP protein family of this species. A phylogenetic analysis of AQPs of Arabidopsis thaliana, B. oleracea, B. rapa allowed us to follow AQP evolution in closely related species and to systematically classify and (re-) name these isoforms. Thirty-three groups of AQP-orthologous genes were identified between B. oleracea and Arabidopsis and their expression was analyzed in different organs. The two selectivity filters, gene structure and coding sequences were highly conserved within each AQP subfamily while sequence variations in some introns and untranslated regions were frequent. These data suggest a similar substrate selectivity and function of Brassica AQPs compared to Arabidopsis orthologs. The comparative analyses of all AQP subfamilies in three Brassicaceae species give initial insights into AQP evolution in these taxa. Based on the genome-wide AQP identification in B. oleracea and the sequence analysis and reprocessing of Brassica AQP information, our dataset provides a sequence resource for further investigations of the physiological and molecular functions of Brassica crop AQPs.