In vitro expression and functional characterization of NPA motifs in aquaporins of Nosema bombycis.

Nosema bombycis contains functional aquaporins (NbAQPs), which are key targets for exploring the mechanism of N. bombycis infection; however, the regulation of these NbAQPs remains unknown. The two highly conserved asparagine-proline-alanine sequences (NPA motifs) play important roles in AQP biogenesis. As part of this study, we constructed a series of NbAQP mutants (NbAQP_NPA1, NbAQP_NPA2, and NbAQP_NPA1,2) and expressed them in BmN cells. The results showed that mutations in either NPA motif or in both NPA motifs did not affect NbAQP expression in vitro. After expression in Xenopus laevis oocytes, those injected with wild-type NbAQP rapidly expanded, whereas oocytes injected with NbAQP_NPAs did not significantly change in size. The associated water permeability (pf) of NbAQP_NPAs was significantly reduced five-six times compared to that of wild-type NbAQP. These results indicated that NPA motifs are necessary for the water channel function of AQPs in N. bombycis. The present study shows for the first time that the NbAQP NPA motif has an impact on the water permeability of aquaporin in N. bombycis, thereby providing a platform for further research into the mechanisms underlying the regulation of NbAQP expression.

Identification of VrNIP2-1 aquaporin with novel selective filter regulating the transport of beneficial as well as hazardous metalloids in mungbean (Vigna radiata L.).

Nodulin 26-like intrinsic protein (NIP) subfamily of aquaporins (AQPs) in plants, is known to be involved in the uptake of metalloids including boron, germanium (Ge), arsenic (As), and silicon (Si). In the present study, a thorough evaluation of 55 AQPs found in the mungbean genome, including phylogenetic distribution, sequence homology, expression profiling, and structural characterization, contributed to the identification of VrNIP2-1 as a metalloid transporter. The pore-morphology of VrNIP2-1 was studied using molecular dynamics simulation. Interestingly, VrNIP2-1 was found to harbor an aromatic/arginine (ar/R) selectivity filter formed with ASGR amino acids instead of GSGR systematically reported in metalloid transporters (NIP2s) in higher plants. Evaluation of diverse cultivars showed a high level of Si accumulation in leaves indicating functional Si transport in mungbean. In addition, heterologous expression of VrNIP2-1 in yeast revealed As(III) and GeO2 transport activity. Similarly, VrNIP2-1 expression in Xenopus oocytes confirmed its Si transport ability. The metalloid transport activity with unique structural features will be helpful to better understand the solute specificity of NIP2s in mungbean and related pulses. The information provided here will also serve as a basis to improve Si uptake while restricting hazardous metalloids like As in plants.

The roles of selectivity filters in determining aluminum transport by AtNIP1;2.

Aquaporins (AQPs) are channel proteins involved in transporting a variety of substrates. It has been proposed that the constriction regions in the central pores of the AQP channels play a crucial role in determining transport substrates and activities of AQPs. Our previous results suggest that AtNIP1;2, a member of the AQP superfamily in Arabidopsis, facilitates aluminum transport across the plasma membrane. However, the functions of the constriction regions in AtNIP1;2-mediated transport activities are unclear. This study reports that residue substitutions of the constriction regions affect AtNIP1;2-mediated aluminum uptake, demonstrating the critical roles of the constriction regions for transport activities. Furthermore, a constriction region that partially or wholly mimics AtNIP5;1, a demonstrated boric-acid transporter, could not render the boric-acid transport activity to AtNIP1;2. Therefore, besides the constriction regions, other structural features are also involved in determining the nature of AtNIP1;2's transport activities.Abbreviations: AIAR: alanine-isoleucine-alanine-arginine; AIGR: alanine-isoleucine-glycine- arginine; AQP: aquaporin; Al-Mal: aluminum-malate; ar/R: aromatic/arginine; AVAR: alanine-valine-alanine-arginine; CK: control; H: helical domain; ICP-MS: inductively coupled plasma mass spectrometry; LA - LE: inter-helical loops A to E; NIP: nodulin 26-like intrinsic protein; NPA: asparagine-proline-alanine; NPG: asparagine-proline- glycine; NPS: asparagine-proline-Serine; NPV: asparagine-proline-valine; ORF: open reading frame; PIP: plasma membrane intrinsic proteins; SIP: small basic intrinsic proteins; TM: transmembrane helices; WIAR: tryptophan-isoleucine-alanine-arginine; WVAR: tryptophan-valine-alanine-arginine; WVGR: tryptophan-valine-glycine- arginine.

Evolutionary Understanding of Aquaporin Transport System in the Basal Eudicot Model Species Aquilegia coerulea.

Aquaporins (AQPs) play a pivotal role in the cellular transport of water and many other small solutes, influencing many physiological and developmental processes in plants. In the present study, extensive bioinformatics analysis of AQPs was performed in Aquilegia coerulea L., a model species belonging to basal eudicots, with a particular focus on understanding the AQPs role in the developing petal nectar spur. A total of 29 AQPs were identified in Aquilegia, and their phylogenetic analysis performed with previously reported AQPs from rice, poplar and Arabidopsis depicted five distinct subfamilies of AQPs. Interestingly, comparative analysis revealed the loss of an uncharacterized intrinsic protein II (XIP-II) group in Aquilegia. The absence of the entire XIP subfamily has been reported in several previous studies, however, the loss of a single clade within the XIP family has not been characterized. Furthermore, protein structure analysis of AQPs was performed to understand pore diversity, which is helpful for the prediction of solute specificity. Similarly, an AQP AqcNIP2-1 was identified in Aquilegia, predicted as a silicon influx transporter based on the presence of features such as the G-S-G-R aromatic arginine selectivity filter, the spacing between asparagine-proline-alanine (NPA) motifs and pore morphology. RNA-seq analysis showed a high expression of tonoplast intrinsic proteins (TIPs) and plasma membrane intrinsic proteins (PIPs) in the developing petal spur. The results presented here will be helpful in understanding the AQP evolution in Aquilegia and their expression regulation, particularly during floral development.