Aromatic Arginine Research Articles

Aquaporin LjNIP1;5 positively modulates drought tolerance by promoting arbuscular mycorrhizal symbiosis in Lotus japonicus

Drought stress often affects crop growth and even causes crop death, while aquaporins can maintain osmotic balance by transporting water across membranes, so it is important to study how to improve drought tolerance of crops by using aquaporins. In this work, we characterize a set of subfamily members named NIPs belonging to the family of aquaporins in Lotus japonicus, grouping 14 family members based on the sequence similarity in the aromatic/arginine (Ar/R) region. Among these members, LjNIP1;5 is one of the genes with the highest expression in roots which is induced by the AM fungus. In Lotus japonicus, LjNIP1;5 is highly expressed in symbiotic roots, and its promoter can be induced by drought stress and AM fungus. Root colonization analysis reveals that ljnip1:5 mutant exhibits lower mycorrhizal colonization than the wild type, with increasing the proportion of large arbuscule, and fewer arbuscule produced by symbiosis under drought stress. In the LjNIP1;5OE plant, we detected a strong antioxidant capacity compared to the control, and LjNIP1;5OE showed higher stem length under drought stress. Taken together, the current results facilitate our comprehensive understanding of the plant adaptive to drought stress with the coordination of the specific fungi.

Significance of solute specificity, expression, and gating mechanism of tonoplast intrinsic protein during development and stress response in plants.

Tonoplast intrinsic proteins (TIPs), belonging to the aquaporin family, are transmembrane channels located mostly at the tonoplast of plant cells. The TIPs are known to transport water and many other small solutes such as ammonia, urea, hydrogen peroxide, and glycerol. In the present review, phylogenetic distribution, structure, transport dynamics, gating mechanism, sub-cellular localization, tissue-specific expression, and co-expression of TIPs are discussed to define their versatile role in plants. Based on the phylogenetic distribution, TIPs are classified into five distinct groups with aromatic-arginine (Ar/R) selectivity filters, typical pore-morphology, and tissue-specific gene expression patterns. The tissue-specific expression of TIPs is conserved among diverse plant species, more particularly for TIP3s, which are expressed exclusively in seeds. Studying TIP3 evolution will help to understand seed development and germination. The solute specificity of TIPs plays an imperative role in physiological processes like stomatal movement and vacuolar sequestration as well as in alleviating environmental stress. TIPs also play an important role in growth and developmental processes like radicle protrusion, anther dehiscence, seed germination, cell elongation, and expansion. The gating mechanism of TIPs regulates the solute flow in response to external signals, which helps to maintain the physiological functions of the cell. The information provided in this review is a base to explore TIP's potential in crop improvement programs.

Characterization of influx and efflux silicon transporters and understanding their role in the osmotic stress tolerance in finger millet (Eleusine coracana (L.) Gaertn.)

Over the last decade, silicon (Si) has been widely accepted as a beneficial element for plant growth. The advantages plant derives from the Si are primarily based on the uptake and transport mechanisms. In the present study, the Si uptake regime was studied in finger millet (Eleusine coracana (L). Gaertn.) under controlled and stress conditions. The finger millet can efficiently uptake Si and accumulate it by more than 1% of dry weight in the leaf tissues, thus categorized as a Si accumulator. Subsequent evaluation with the single root assay revealed a three-fold higher Si uptake under osmatic stress than control. These results suggest that Si alleviated the PEG-induced stress by regulating the levels of osmolytes and antioxidant enzymes. Further, to understand the molecular mechanism involved in Si uptake, the Si influx (EcoLsi1 and EcoLsi6) and efflux transporters (EcoLsi2 and EcoLsi3) were identified and characterized. The comparative phylogenomic analysis of the influx transporter EcoLsi1 with other monocots revealed conserved features like aromatic/arginine (Ar/R) selectivity filters and pore morphology. Similarly, Si efflux transporter EcoLsi3 is highly homologous to other annotated efflux transporters. The transcriptome data revealed that the expression of both influx and efflux Si transporters was elevated due to Si supplementation under stress conditions. These findings suggest that stress elevates Si uptake in finger millet, and its transport is also regulated by the Si transporters. The present study will be helpful to better explore Si derived benefits in finger millet.

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.

Presence of Intra-helical Salt-Bridge in Loop E Half-Helix Can Influence the Transport Properties of AQP1 and GlpF Channels: Molecular Dynamics Simulations of In Silico Mutants.

Major intrinsic protein (MIP) superfamily contains water-transporting AQP1 and glycerol-specific GlpF belonging to two major phylogenetic groups, namely aquaporins (AQPs) and aquaglyceroporins (AQGPs). MIP channels have six transmembrane helices (TM1 to TM6) and two half-helices (LB and LE). LE region contributes two residues to the aromatic/arginine (Ar/R) selectivity filter (SF) within the MIP channel. Bioinformatics analyses have shown that all AQGPs have an intra-helical salt-bridge (IHSB) in LE half-helix and all AQGPs and majority of AQPs have helix destabilizing Gly and/or Pro in the same region. In this paper, we mutated in silico the acidic and basic residues in GlpF to Ser and introduced salt-bridge interaction in AQP1 LE half-helix by substituting Ser residues at the equivalent positions with acidic and basic residues. We investigated the influence of IHSB in LE half-helix on the transport properties of GlpF and AQP1 mutant channels using molecular dynamics simulations. With IHSB abolished in LE half-helix, the GlpF mutant exhibited a significantly reduced water transport. In contrast, the introduction of IHSB in the two AQP1 mutants has increased water transport. Absence of salt-bridge in LE half-helix alters the SF geometry and results in a higher energy barrier for the solutes in the Ar/R selectivity filter. Presence/absence of IHSB in LE half-helix influences the channel transport properties and it is evident especially for the AQGPs. By modulating its helical flexibility, LE half-helix can perhaps play a regulatory role in transport either on its own or in conjunction with other extracellular regions.

Facilitated Water Transport through Graphene Oxide Membranes Functionalized with Aquaporin-Mimicking Peptides

Water purification by membranes is widely investigated to address concerns related to clean water scarcity. Achieving high flux and rejection simultaneously is a difficult challenge using such membranes because these properties are mutually exclusive in common artificial membranes. Nature has developed a method for this task involving water-channel membrane proteins known as aquaporins. Here, the design and fabrication of graphene oxide (GO)-based membranes with a surface-tethered peptide motif designed to mimic the water-selective filter of natural aquaporins is reported. The short RF8 (RFRFRFRF, where R and F represent arginine and phenylalanine, respectively) octapeptide is a concentrated form of the core component of the Ar/R (aromatic/arginine) water-selective filter in aquaporin. The resulting GO-RF8 shows superior flux and high rejection similar to natural quaporins. Molecular dynamics simulation revealed the unique configuration of RF8 peptides and the transport of water in GO-RF8 membranes, supporting that RF8 effectively emulates the core function of aquaporins.

Facilitated Water Transport through Graphene Oxide Membranes Functionalized with Aquaporin‐Mimicking Peptides

Water purification by membranes is widely investigated to address concerns related to the scarcity of clean water. Achieving high flux and rejection simultaneously is a difficult challenge using such membranes because these properties are mutually exclusive in common artificial membranes. Nature has developed a method for this task involving water-channel membrane proteins known as aquaporins. Here, the design and fabrication of graphene oxide (GO)-based membranes with a surface-tethered peptide motif designed to mimic the water-selective filter of natural aquaporins is reported. The short RF8 (RFRFRFRF, where R and F represent arginine and phenylalanine, respectively) octapeptide is a concentrated form of the core component of the Ar/R (aromatic/arginine) water-selective filter in aquaporin. The resulting GO-RF8 shows superior flux and high rejection similar to natural aquaporins. Molecular dynamics simulation reveal the unique configuration of RF8 peptides and the transport of water in GO-RF8 membranes, supporting that RF8 effectively emulates the core function of aquaporins.

Genome-wide identification, characterization, and expression profile of aquaporin gene family in flax (Linum usitatissimum)

Membrane intrinsic proteins (MIPs) form transmembrane channels and facilitate transport of myriad substrates across the cell membrane in many organisms. Majority of plant MIPs have water transporting ability and are commonly referred as aquaporins (AQPs). In the present study, we identified aquaporin coding genes in flax by genome-wide analysis, their structure, function and expression pattern by pan-genome exploration. Cross-genera phylogenetic analysis with known aquaporins from rice, arabidopsis, and poplar showed five subgroups of flax aquaporins representing 16 plasma membrane intrinsic proteins (PIPs), 17 tonoplast intrinsic proteins (TIPs), 13 NOD26-like intrinsic proteins (NIPs), 2 small basic intrinsic proteins (SIPs), and 3 uncharacterized intrinsic proteins (XIPs). Amongst aquaporins, PIPs contained hydrophilic aromatic arginine (ar/R) selective filter but TIP, NIP, SIP and XIP subfamilies mostly contained hydrophobic ar/R selective filter. Analysis of RNA-seq and microarray data revealed high expression of PIPs in multiple tissues, low expression of NIPs, and seed specific expression of TIP3 in flax. Exploration of aquaporin homologs in three closely related Linum species bienne, grandiflorum and leonii revealed presence of 49, 39 and 19 AQPs, respectively. The genome-wide identification of aquaporins, first in flax, provides insight to elucidate their physiological and developmental roles in flax.

Structure and Dynamics of Extracellular Loops in Human Aquaporin-1 from Solid-State NMR and Molecular Dynamics.

Multiple moderate-resolution crystal structures of human aquaporin-1 have provided a foundation for understanding the molecular mechanism of selective water translocation in human cells. To gain insight into the interfacial structure and dynamics of human aquaporin-1 in a lipid environment, we performed nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations. Using magic angle spinning solid-state NMR, we report a near complete resonance assignment of the human aquaporin-1. Chemical shift analysis of the secondary structure identified pronounced deviations from crystallographic structures in extracellular loops A and C, including the cis Y37-P38 bond in loop A, as well as ordering and immobilization of loop C. Site-specific H/D exchange measurements identify a number of protected nitrogen-bearing side chains and backbone amide groups, involved in stabilizing the loops. A combination of molecular dynamics simulations with NMR-derived restraints and filtering based on solvent accessibility allowed for the determination of a structural model of extracellular loops largely consistent with NMR results. The simulations reveal loop stabilizing interactions that alter the extracellular surface of human AQP1, with possible implications for water transport regulation through the channel. Modulation of water permeation may occur as a result of rearrangement of side chains from loop C in the extracellular vestibule of hAQP1, affecting the aromatic arginine selectivity filter.

An aquaporin PvTIP4;1 from Pteris vittata may mediate arsenite uptake.

The fern Pteris vittata is an arsenic hyperaccumulator. The genes involved in arsenite (As(III)) transport are not yet clear. Here, we describe the isolation and characterization of a new P.vittata aquaporin gene, PvTIP4;1, which may mediate As(III) uptake. PvTIP4;1 was identified from yeast functional complement cDNA library of P.vittata. Arsenic toxicity and accumulating activities of PvTIP4;1 were analyzed in Saccharomyces cerevisiae and Arabidopsis. Subcellular localization of PvTIP4;1-GFP fusion protein in P.vittata protoplast and callus was conducted. The tissue expression of PvTIP4;1 was investigated by quantitative real-time PCR. Site-directed mutagenesis of the PvTIP4;1 aromatic/arginine (Ar/R) domain was studied. Heterologous expression in yeast demonstrates that PvTIP4;1 was able to facilitate As(III) diffusion. Transgenic Arabidopsis showed that PvTIP4;1 increases arsenic accumulation and induces arsenic sensitivity. Images and FM4-64 staining suggest that PvTIP4;1 localizes to the plasma membrane in P.vittata cells. A tissue location study shows that PvTIP4;1 transcripts are mainly expressed in roots. Site-directed mutation in yeast further proved that the cysteine at the LE1 position of PvTIP4;1 Ar/R domain is a functional site. PvTIP4;1 is a new represented tonoplast intrinsic protein (TIP) aquaporin from P.vittata and the function and location results imply that PvTIP4;1 may be involved in As(III) uptake.

Making an Aquaporin Water-Tight: Structural Basis of Selectivity in Plant Nodulin 26 Intrinsic Proteins

The evolution of land plants led toan amplification and diversification of the aquaporin superfamily of membrane channels. Among the subfamilies of plant specific aquaporin-like changes are the nodulin-26 intrinsic proteins (NIPs) which are multifunctional transporters of water, ammonia, glycerol and metalloid nutrients that participate in a number of osmoregulatory and metabolic functions. NIPs share the canonical hourglass fold of the aquaporin family, but possess substitutions within the aromatic arginine (ar/R) selectivity filter. The nine proteins of the NIP subfamily in the model plant Arabidopsis thaliana can be subdivided into two ar/R pore subgroups: the NIP subgroup I, which form aquaglyceroporins that are permeated by glycerol, water and ammonia, and the NIP subgroup II, which form metalloid transporters which are lack aquaporin activity and are essentially “water tight”. These two NIP pore families differ principally by the substitution of a conserved alanine (NIP subgroup II) for a conserved tryptophan (NIP subgroup I) in the helix 2 position (H2) of the ar/R filter. Based on transport analyses and molecular dynamics simulation, a model is proposed through which the alanine substitution results in both the selectivity for critical metalloid nutrients such as boric acid while simultaneously restricting water flow through the ar/R selectivity filter. A mechanism involving two different rotameric states of the conserved arginine residue in this selectivity region is proposed to be responsible for the water-tight character of the pore. (Supported in part by NSF grant 1121465).

Arabidopsis thalianaNIP7;1: An Anther-Specific Boric Acid Transporter of the Aquaporin Superfamily Regulated by an Unusual Tyrosine in Helix 2 of the Transport Pore

Plant nodulin-26 intrinsic proteins (NIPs) are members of the aquaporin superfamily that serve as multifunctional transporters of uncharged metabolites. In Arabidopsis thaliana, a specific NIP pore subclass, known as the NIP II proteins, is represented by AtNIP5;1 and AtNIP6;1, which encode channel proteins expressed in roots and leaf nodes, respectively, that participate in the transport of the critical cell wall nutrient boric acid. Modeling of the protein encoded by the AtNIP7;1 gene shows that it is a third member of the NIP II pore subclass in Arabidopsis. However, unlike AtNIP5;1 and AtNIP6;1 proteins, which form constitutive boric acid channels, AtNIP7;1 forms a channel with an extremely low intrinsic boric acid transport activity. Molecular modeling and molecular dynamics simulations of AtNIP7;1 suggest that a conserved tyrosine residue (Tyr81) located in transmembrane helix 2 adjacent to the aromatic arginine (ar/R) pore selectivity region stabilizes a closed pore conformation through interaction with the canonical Arg220 in ar/R region. Substitution of Tyr81 with a Cys residue, characteristic of established NIP boric acid channels, results in opening of the AtNIP7;1 pore that acquires a robust, transport activity for boric acid as well as other NIP II test solutes (glycerol and urea). Substitution of a Phe for Tyr81 also opens the channel, supporting the prediction from MD simulations that hydrogen bond interaction between the Tyr81 phenol group and the ar/R Arg may contribute to the stabilization of a closed pore state. Expression analyses show that AtNIP7;1 is selectively expressed in developing anther tissues of young floral buds of A. thaliana, principally in developing pollen grains of stage 9-11 anthers. Because boric acid is both an essential nutrient as well as a toxic compound at high concentrations, it is proposed that Tyr81 modulates transport and may provide an additional level of regulation for this transporter in male gametophyte development.