Intrinsic Membrane Proteins Research Articles

Aquaporins Responses under Challenging Environmental Conditions and Abiotic Stress Tolerance in Plants

Aquaporins are important transmembrane channel proteins required for the transport of water and nutrients to maintain plant water relations and its survival. The aquaporins have a characteristic structure and are divided into 5 distinct isoforms such as plasma membrane intrinsic proteins (PIP), tonoplast intrinsic protein (TIP), small basic intrinsic protein (SIP), nodulin-26 like intrinsic protein (NIP) and X intrinsic proteins (XIP). Aquaporins conducts transcellular movement of water molecules across the cell and organelles membranes. Apart from playing key role in maintaining water level inside the plant system it is also involved in transporting small neutral molecules including glycerol, urea, metalloids, carbon dioxide, nitrogen, hydrogen peroxide and ammonia. In this way it regulates ionic homeostasis and water levels during environmental stresses including drought, chilling, salinity, and heavy metal toxicity and helps in providing tolerance toward the stress. It also plays a key role in maintaining the carbon and nitrogen level in the cell, transporting the key regulatory molecules and phytohormones mediated tolerance to plants. Scientist has also targeted aquaporins in order to develop stress tolerant transgenic species having improved functioning of aquaporins. Increase of water retention capability and usage efficiency by the plants represents the major research areas to confer abiotic stress tolerance. This review presents a comprehensive studies and intensive ongoing research to understand the diverse role of aquaporins in ensuring normal growth and development of plants. Current aspects of aquaporins and its isoforms in relation to its structure, function and regulation in different plant species have been highlighted. Further the potential of aquaporins in promoting plant survival during wide ranges of environmental situations is also emphasized.

SLCO1B1: Application and Limitations of Deep Mutational Scanning for Genomic Missense Variant Function.

SLCO1B1 (solute carrier organic anion transporter family member 1B1) is an important transmembrane hepatic uptake transporter. Genetic variants in the SLCO1B1 gene have been associated with altered protein folding, resulting in protein degradation and decreased transporter activity. Next-generation sequencing (NGS) of pharmacogenes is being applied increasingly to associate variation in drug response with genetic sequence variants. However, it is difficult to link variants of unknown significance with functional phenotypes using “one-at-a-time” functional systems. Deep mutational scanning (DMS) using a “landing pad cell–based system” is a high-throughput technique designed to analyze hundreds of gene open reading frame (ORF) missense variants in a parallel and scalable fashion. We have applied DMS to analyze 137 missense variants in the SLCO1B1 ORF obtained from the Exome Aggregation Consortium project. ORFs containing these variants were fused to green fluorescent protein and were integrated into “landing pad” cells. Florescence-activated cell sorting was performed to separate the cells into four groups based on fluorescence readout indicating protein expression at the single cell level. NGS was then performed and SLCO1B1 variant frequencies were used to determine protein abundance. We found that six variants not previously characterized functionally displayed less than 25% and another 12 displayed approximately 50% of wild-type protein expression. These results were then functionally validated by transporter studies. Severely damaging variants identified by DMS may have clinical relevance for SLCO1B1-dependent drug transport, but we need to exercise caution since the relatively small number of severely damaging variants identified raise questions with regard to the application of DMS to intrinsic membrane proteins such as organic anion transporter protein 1B1.Significance StatementThe functional implications of a large numbers of open reading frame (ORF) “variants of unknown significance” (VUS) in transporter genes have not been characterized. This study applied deep mutational scanning to determine the functional effects of VUS that have been observed in the ORF of SLCO1B1(solute carrier organic anion transporter family member 1B1). Several severely damaging variants were identified, studied, and validated. These observations have implications for both the application of deep mutational scanning to intrinsic membrane proteins and for the clinical effect of drugs and endogenous compounds transported by SLCO1B1.

Mycorrhizal response strategies of trifoliate orange under well-watered, salt stress, and waterlogging stress by regulating leaf aquaporin expression

Aquaporins (AQPs) involved in water and small molecule transport respond to environmental stress, while it is not clear how arbuscular mycorrhizal fungi (AMF) regulate AQP expression. Here, we investigated the change in leaf water potential and expression level of four tonoplast intrinsic proteins (TIPs), six plasma membrane intrinsic proteins (PIPs), and four nodin-26 like intrinsic proteins (NIPs) genes in trifoliate orange (Poncirus trifoliata) inoculated with Funneliformis mosseae under well-watered (WW), salt stress (SS), and waterlogging stress (WS). Root AMF colonization and soil hyphal length collectively were reduced by SS and WS. Under WW, inoculation with AMF gave diverse responses of AQPs: six AQPs up-regulated, three AQPs down-regulated, and five AQPs did not change. Such up-regulation of more AQPs under mycorrhization and WW partly accelerated water absorption, thereby, maintaining higher leaf water potential. However, under SS, all the fourteen AQPs were dramatically induced by AMF inoculation, which improved water permeability of membranes and stimulated water transport of the host. Under WS, AMF colonization almost did not induce or even down-regulated these AQPs expressions with three exceptions (PtTIP2;2, PtPIP1;1, and PtNIP1;2), thus, no change in leaf water potential. As a result, mycorrhizal plants under flooding may have an escape mechanism to reduce water absorption. It is concluded that AMF had different strategies in response to environmental stresses (e.g. SS and WS) by regulating leaf AQP expression in the host (e.g. trifoliate orange).

The pip1s Quintuple Mutants Demonstrate the Essential Roles of PIP1s in the Plant Growth and Development of Arabidopsis.

Plasma membrane intrinsic proteins (PIPs) transport water, CO2 and small neutral solutes across the plasma membranes. In this study, we used the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 system (CRISPR/Cas9) to mutate PIP1;4 and PIP1;5 in a pip1;1,2,3 triple mutant to generate a pip1;1,2,3,4,5 (pip1s−) quintuple mutant. Compared to the wild-type (WT) plant, the pip1s− mutants had smaller sized rosette leaves and flowers, less rosette leaf number, more undeveloped siliques, shorter silique and less seeds. The pollen germination rate of the pip1s− mutant was significantly lower than that of the WT and the outer wall of the pip1s− mutant’s pollen was deformed. The transcriptomic analysis showed significant alterations in the expression of many key genes and transcription factors (TFs) in the pip1s− mutant which involved in the development of leaf, flower and pollen, suggesting that the mutant of PIP1s not only directly affects hydraulics and carbon fixation, but also regulates the expression of related genes to affect plant growth and development.

Decreases in arsenic accumulation by the plasma membrane intrinsic protein PIP2;2 in Arabidopsis and yeast.

Arsenic (As) is a toxic pollutant that mainly enters the human body via plants. Therefore, understanding the strategy for reducing arsenic accumulation in plants is important to human health and the environment. Aquaporins are ubiquitous water channel proteins that bidirectionally transport water across cell membranes and play a role in the transportation of other molecules, such as glycerol, ammonia, boric acid, and arsenic acid. Previously, we observed that Arabidopsis PIP2;2, encoding a plasma membrane intrinsic protein, is highly expressed in NtCyc07-expressing Arabidopsis, which shows a higher tolerance to arsenite (As(III)). In this study, we report that the overexpression of AtPIP2;2 enhanced As(III) tolerance and reduced As(III) levels in yeast. Likewise, AtPIP2;2-overexpressing Arabidopsis exhibited improved As(III) tolerance and lower accumulation of As(III). In contrast, atpip2;2 knockout Arabidopsis showed reduced As(III) tolerance but no significant change in As(III) levels. Interestingly, the AtPIP2;2 transcript and protein levels were increased in roots and shoots of Arabidopsis in response to As(III). Furthermore, As(III) efflux was enhanced and As(III) influx/accumulation was reduced in AtPIP2;2-expressing plants. The expression of AtPIP2;2 rescued the As(III)-sensitive phenotype of acr3 mutant yeast by reducing As levels and slightly reduced the As(III)-tolerant phenotype of fps1 mutant yeast by enhancing As content, suggesting that AtPIP2; 2 functions as a bidirectional channel of As(III), while the As(III) exporter activity is higher than the As(III) importer activity. All these results indicate that AtPIP2;2 expression promotes As(III) tolerance by decreasing As(III) accumulation through enhancing As(III) efflux in Arabidopsis. This finding can be applied to the generation of low arsenic crops for human health.

Response of spikelet water status to high temperature and its relationship with heat tolerance in rice

In rice, high-temperature stress (HT) during flowering results in decreased grain yield via a reduction in spikelet fertility; however, the effect of plant water status on spikelet fertility under HT remains unknown. To investigate the relationship between spikelet water status and spikelet fertility under HT, two experiments were performed under temperature-controlled conditions using four genotypes with varying tolerance to HT. Rice plants were exposed to HT for seven consecutive days during the flowering stage under three soil water treatments (soil water potential 0, −20, and −40 kPa), as well as under hydroponic conditions in a separate experiment. HT significantly decreased spikelet fertility, pollen fertility, and anther dehiscence under each of the three water treatments. HT significantly increased the spikelet transpiration rate, and this change was accompanied by a significant decrease in the internal temperature of the spikelets. HT decreased pollen grain diameter in heat-sensitive genotypes. HT had varying effects on the water potential of panicles and anthers but increased anther soluble-sugar concentration. Different aquaporin genes showed different expression profiles under HT, and the expression levels of PIPs for plasma membrane intrinsic proteins and TIPs for tonoplast intrinsic proteins increased in anthers but decreased in glumes. Correlation analyses showed that anther dehiscence and pollen (spikelet) fertility were tightly associated with anther water status, and the expression levels of almost all anther aquaporin genes were significantly correlated with anther dehiscence under HT. In summary, an increased spikelet transpiration rate and decreased internal spikelet temperature were associated with alleviation of the effects of HT in rice genotypes with varying degrees of heat tolerance, and the response of spikelet water status to HT, involving increased total expression of aquaporins and soluble sugar content, thereby improved pollen fertility, anther dehiscence, and spikelet fertility, especially in heat-resistant genotypes. The heat-resistant genotypes N22 and SY63 may adopt different approaches to reduce heat damage.

Tocopherol controls D1 amino acid oxidation by oxygen radicals in Photosystem II

Photosystem II (PSII) is an intrinsic membrane protein complex that functions as a light-driven water:plastoquinone oxidoreductase in oxygenic photosynthesis. Electron transport in PSII is associated with formation of reactive oxygen species (ROS) responsible for oxidative modifications of PSII proteins. In this study, oxidative modifications of the D1 and D2 proteins by the superoxide anion (O2•-) and the hydroxyl (HO•) radicals were studied in WT and a tocopherol cyclase (vte1) mutant, which is deficient in the lipid-soluble antioxidant α-tocopherol. In the absence of this antioxidant, high-resolution tandem mass spectrometry was used to identify oxidation of D1:130E to hydroxyglutamic acid by O2•- at the PheoD1 site. Additionally, D1:246Y was modified to either tyrosine hydroperoxide or dihydroxyphenylalanine by O2•- and HO•, respectively, in the vicinity of the nonheme iron. We propose that α-tocopherol is localized near PheoD1 and the nonheme iron, with its chromanol head exposed to the lipid-water interface. This helps to prevent oxidative modification of the amino acid's hydrogen that is bonded to PheoD1 and the nonheme iron (via bicarbonate), and thus protects electron transport in PSII from ROS damage.

Interaction Between the SNARE SYP121 and the Plasma Membrane Aquaporin PIP2;7 Involves Different Protein Domains.

Plasma membrane intrinsic proteins (PIPs) are channels facilitating the passive diffusion of water and small solutes. Arabidopsis PIP2;7 trafficking occurs through physical interaction with SNARE proteins including the syntaxin SYP121, a plasma membrane Qa-SNARE involved in membrane fusion. To better understand the interaction mechanism, we aimed at identifying the interaction motifs in SYP121 and PIP2;7 using ratiometric bimolecular fluorescence complementation assays in Nicotiana benthamiana. SYP121 consists of four regions, N, H, Q, and C, and sequential deletions revealed that the C region, containing the transmembrane domain, as well as the H and Q regions, containing the Habc and Qa-SNARE functional domains, interact with PIP2;7. Neither the linker between the Habc and the Qa-SNARE domains nor the H or Q regions alone could fully restore the interaction with PIP2;7, suggesting that the interacting motif depends on the conformation taken by the HQ region. When investigating the interacting motif(s) in PIP2;7, we observed that deletion of the cytosolic N- and/or C- terminus led to a significant decrease in the interaction with SYP121. Shorter deletions revealed that at the N-terminal amino acid residues 18–26 were involved in the interaction. Domain swapping experiments between PIP2;7 and PIP2;6, a PIP isoform that does not interact with SYP121, showed that PIP2;7 N-terminal part up to the loop C was required to restore the full interaction signal, suggesting that, as it is the case for SYP121, the interaction motif(s) in PIP2;7 depend on the protein conformation. Finally, we also showed that PIP2;7 physically interacted with other Arabidopsis SYP1s and SYP121 orthologs.

Polyamine homeostasis modulates plasma membrane- and tonoplast-associated aquaporin expression in etiolated salt-stressed sunflower (Helianthus annuus L.) seedlings.

Salt stress adversely affects plants by causing osmotic and ionic imbalance. Cellular osmotic adjustment occurs by modulation of water fluxes. Polyamines (PAs) are often advocated to be involved in osmoregulation during stressful conditions, and thus, they serve as potential "osmolytes." Aquaporins (AQPs), the water-transporting channels, are expected to play crucial roles in osmoregulation. Present investigations on etiolated sunflower seedlings demonstrate a possible correlation between PA homeostasis and maintenance of water balance, as a function of modulation of the abundance of two major AQP subfamilies: PIP2 (plasma membrane intrinsic protein 2) and TIP1 (tonoplast intrinsic protein 1). Salt stress (120 mM NaCl) restricts growth of sunflower seedlings and induces reduction in relative water content (RWC). This accompanies enhanced abundance of PIP2s and TIP1s in seedling roots and that of TIP1s in cotyledons, as revealed by Western blot analysis of AQP isoforms and also their imaging by confocal laser scanning microscopy (CLSM). Raising seedlings in the presence of 500 μM of DFMA (DL-α-difluoromethylarginine) or DFMO (DL-α-difluoromethylornithine), which are potent inhibitors of PA biosynthesis enzymes (arginine decarboxylase (ADC) and ornithine decarboxylase (ODC), respectively), significantly promotes root extension, irrespective of NaCl stress, and results in further lowering of salt-induced reduction in RWC in roots and cotyledons. This correlates with enhanced accumulation of both PIP2s and TIP1s in seedling roots, but not in cotyledons. Present work, therefore, implicates PA homeostasis in the maintenance of water status of sunflower seedlings, possibly via regulation of abundance and distribution of AQP isoforms associated with the plasma membrane and tonoplast.

A native cell membrane nanoparticles system allows for high-quality functional proteoliposome reconstitution

Proteoliposomes mimic the cell membrane environment allowing for structural and functional membrane protein analyses as well as antigen presenting and drug delivery devices. To make proteoliposomes, purified functional membrane proteins are required. Detergents have traditionally been used for the first step in this process. However, they can irreversibly denature or render membrane proteins unstable, and the necessary removal of detergents after reconstitution can decrease proteoliposome yields. The recently developed native cell membrane nanoparticles (NCMN) system has provided a variety of detergent-free alternatives for membrane protein preparation for structural biology research. Here we attempt to employ the MCMN system for the functional reconstitution of channels into proteoliposomes. NCMN polymers NCMNP1-1 and NCMNP7-1, members of a NCMN polymer library that have been successful in extraction and affinity purification of a number of intrinsic membrane proteins, were selected for the purification and subsequent reconstitution of three bacterial channels: KcsA and the mechanosensitive channels of large and small conductance (MscL and MscS). We found that channels in NCMN particles, which appeared to be remarkably stable when stored at 4 °C, can be reconstituted into bilayers by simply incubating with lipids. We show that the resulting proteoliposomes can be patched for electrophysiological studies or used for the generation of liposome-based nanodevices. In sum, the findings demonstrate that the NCMN system is a simple and robust membrane protein extraction and reconstitution approach for making high-quality functional proteoliposomes that could significantly impact membrane protein research and the development of nanodevices.

Controversial Regulation of Gene Expression and Protein Transduction of Aquaporins under Drought and Salinity Stress.

Enhancement of the passage of water through membranes is one of the main mechanisms via which cells can maintain their homeostasis under stress conditions, and aquaporins are the main participants in this process. However, in the last few years, a number of studies have reported discrepancies between aquaporin messenger RNA (mRNA) expression and the number of aquaporin proteins synthesised in response to abiotic stress. These observations suggest the existence of post-transcriptional mechanisms which regulate plasma membrane intrinsic protein (PIP) trafficking to the plasma membrane. This indicates that the mRNA synthesis of some aquaporins could be modulated by the accumulation of the corresponding encoded protein, in relation to the turnover of the membranes. This aspect is discussed in terms of the results obtained: on the one hand, with isolated vesicles, in which the level of proteins present provides the membranes with important characteristics such as resistance and stability and, on the other, with isolated proteins reconstituted in artificial liposomes as an in vitro method to address the in vivo physiology of the entire plant.

Isolation, Cloning and Characterization of a Constitutive Plant from Potato Aquaporin Gene

Plasma membrane intrinsic proteins (PIP1) are the most common integral membrane proteins belong to a larger family of intrinsic aquaporin proteins. They are member of aquaporin gene family and have gained importance as highly expressed genes in plants. In this study, the promoter of aquaporin PIP1 gene was identified, analyzed and retrieved from high throughput genomic sequence (HTGS) database. The cis-acting regulatory elements, transcription start sites and transcription factor binding sites of selected promoter were identified through different bio-informatics tools. Many light responsive, phytohormone, stress and defense related cis-regulatory elements were detected in PIP1 promoter region indicating its role as a constitutive promoter. The PIP1 promoter was isolated from Solanum tuberosum. It was initially cloned in TA vector (pTZ57R/T) and later transferred to plant expression binary vectors, pGR1 and pGA482 for transient and stable expression studies in tobacco. The GUS expression results of PIP1 promoter in different tobacco tissues showed its functional importance in regulating gene expression in a constitutive manner. Further, it was concluded that the PIP1 aquaporin promoter is constitutively expressed with a strength equivalent to CaMV 2x35S promoter. These findings indicated the significance of isolated promoter for genetic engineering of plants for crop improvement.

Mechanisms Activating Latent Functions of PIP Aquaporin Water Channels via the Interaction between PIP1 and PIP2 Proteins.

Plant plasma membrane-type plasma membrane intrinsic protein (PIP) aquaporins are classified into two groups, PIP1s and PIP2s. In this study, we focused on HvPIP1;2, a PIP1 in barley (Hordeum vulgare), to dissect the molecular mechanisms that evoke HvPIP1-mediated water transport. No HvPIP1;2 protein was localized to the plasma membrane when expressed alone in Xenopus laevis oocytes. By contrast, a chimeric HvPIP1;2 protein (HvPIP1;2_24NC), in which the N- and C-terminal regions were replaced with the corresponding regions from HvPIP2;4, was found to localize to the plasma membrane of oocytes. However, HvPIP1;2_24NC showed no water transport activity in swelling assays. These results suggested that the terminal regions of PIP2 proteins direct PIP proteins to the plasma membrane, but the relocalization of PIP1 proteins was not sufficient to PIP1s functionality as a water channel in a membrane. A single amino acid replacement of threonine by methionine in HvPIP2;4 (HvPIP2;4T229M) abolished water transport activity. Co-expression of HvPIP1;2_24NC either with HvPIP2;4_12NC or with HvPIP2;4TM_12NC, in which the N- and C-terminal regions were replaced with the corresponding regions of HvPIP1;2, increased the water transport activity in oocytes. These data provided evidence that the HvPIP1;2 molecule has own water transport activity and an interaction with the middle part of the HvPIP2;4 protein (except for the N- and C-termini) is required for HvPIP1;2 functionality as a water channel. This molecular mechanism could be applied to other PIP1s and PIP2s in addition to the known mechanism that the terminal regions of some PIP2s lead some PIP1s to the plasma membrane.

Nef homodimers down-regulate SERINC5 by AP-2–mediated endocytosis to promote HIV-1 infectivity

SERINC5 is a multipass intrinsic membrane protein that suppresses HIV-1 infectivity when incorporated into budding virions. The HIV-1 Nef virulence factor prevents viral incorporation of SERINC5 by triggering its down-regulation from the producer cell membrane through an AP-2-dependent endolysosomal pathway. However, the mechanistic basis for SERINC5 down-regulation by Nef remains elusive. Here we demonstrate that Nef homodimers are important for SERINC5 down-regulation, trafficking to late endosomes, and exclusion from newly synthesized viral particles. Based on previous X-ray crystal structures, we mutated three conserved residues in the Nef dimer interface (Leu112, Tyr115, and Phe121) and demonstrated attenuated homodimer formation in a cell-based fluorescence complementation assay. Point mutations at each position reduced the infectivity of HIV-1 produced from transfected 293T cells, the Jurkat TAg T-cell line, and donor mononuclear cells in a SERINC5-dependent manner. In SERINC5-transfected 293T cells, virion incorporation of SERINC5 was increased by dimerization-defective Nef mutants, whereas down-regulation of SERINC5 from the membrane of transfected Jurkat cells by these mutants was significantly reduced. Nef dimer interface mutants also failed to trigger internalization of SERINC5 and localization to Rab7+ late endosomes in T cells. Importantly, fluorescence complementation assays demonstrated that dimerization-defective Nef mutants retained interaction with both SERINC5 and AP-2. These results show that down-regulation of SERINC5 and subsequent enhancement of viral infectivity require Nef homodimers and support a mechanism by which the Nef dimer bridges SERINC5 to AP-2 for endocytosis. Pharmacological disruption of Nef homodimers may control HIV-1 infectivity and viral spread by enhancing virion incorporation of SERINC5.

Functional analysis of plasma membrane intrinsic protein ZmPIP1;1 involved in drought tolerance and photosynthesis in maize

<p indent=0mm>Plasma membrane intrinsic protein (PIP) is one of the main subfamily of aquaporin regulates diverse physiology functions during plant growth and development. In previous research, the expression of <italic>ZmPIP1</italic>;<italic>1</italic> was induced by osmosis or salt stress. However, the biological function of <italic>ZmPIP1</italic>;<italic>1</italic> was still unclear in maize. In this study, <italic>ZmPIP1</italic>;<italic>1</italic> overexpressed transgenic plants were obtained and exhibited less water loss rate and enhanced drought tolerance compared to wild type. Transcriptome sequencing indicated significant changes in the expression levels of genes involved in ABA biosynthesis and its signaling pathways. Photosynthetic activity, kernel width and kernel weight was increased in <italic>ZmPIP1</italic>;<italic>1</italic> overexpression maize, but not growth under normal condition compared with wild type. Moreover, interaction between ZmPIP1;1 and ZmPIP2;6 was observed by bimolecular fluorescence complementation (BiFC) experiment, resulting in re-localized on plasma membrane and chloroplast in maize mesophyll protoplast. Our study laid an important foundation for understanding the molecular mechanism of <italic>ZmPIP1</italic>;<italic>1</italic>, and provided a new method of molecular breeding for high photosynthetic efficiency.

Aquaporins are main contributors to root hydraulic conductivity in pearl millet [Pennisetum glaucum (L) R. Br.

Pearl millet is a key cereal for food security in arid and semi-arid regions but its yield is increasingly threatened by water stress. Physiological mechanisms relating to conservation of soil water or increased water use efficiency can alleviate that stress. Aquaporins (AQP) are water channels that mediate root water transport, thereby influencing plant hydraulics, transpiration and soil water conservation. However, AQP remain largely uncharacterized in pearl millet. Here, we studied AQP function in root water transport in two pearl millet lines contrasting for water use efficiency (WUE). We observed that these lines also contrasted for root hydraulic conductivity (Lpr) and AQP contribution to Lpr. The line with lower WUE showed significantly higher AQP contribution to Lpr. To investigate AQP isoforms contributing to Lpr, we developed genomic approaches to first identify the entire AQP family in pearl millet and secondly, characterize the plasma membrane intrinsic proteins (PIP) gene expression profile. We identified and annotated 33 AQP genes in pearl millet, among which ten encoded PIP isoforms. PgPIP1-3 and PgPIP1-4 were significantly more expressed in the line showing lower WUE, higher Lpr and higher AQP contribution to Lpr. Overall, our study suggests that the PIP1 AQP family are the main regulators of Lpr in pearl millet and may possibly be associated with mechanisms associated to whole plant water use. This study paves the way for further investigations on AQP functions in pearl millet hydraulics and adaptation to environmental stresses.

Understanding aquaporin transport system in highly stress-tolerant and medicinal plant species Jujube (Ziziphus jujuba Mill.)

Jujube (Ziziphus jujubaMill.), a deciduous tree, is well known for its medicinal and nutritional values. Being an extremophile, it has an excellent capability to survive under arid conditions with limited water availability. In this regard, studying the role of water transport regulating proteins such as Aquaporins (AQPs) in jujube is of great importance. Aquaporins, channel-forming proteins are known to have a significant role in the transport of water and many other small solutes in plants. In the present study, computational approaches have identified 36 AQPs, which comprised of 12 NIPs (Nodulin 26-like intrinsic proteins), 10 PIPs (Plasma membrane intrinsic proteins), 10 TIPs (Tonoplast intrinsic proteins), 3 SIPs (Small intrinsic proteins), and 1 XIP (uncharacterized intrinsic protein). Conserved features of AQPs like asparagines-proline-alanine (NPA) amino acid motifs, aromatic/arginine (ar/R) selectivity filters, and Frogger's residues, having a significant role in solute specificity and transport, were also predicted. Homology-based tertiary (3D) structures of AQPS were also resolved using various tools, and subsequently, pore-lining residues have been identified using the 3D structures. The information of pore morphology, along with the conserved features provided through this work, will be helpful to predict solute specificity of AQPs. Analysis of transcriptomic data revealed the tissue-specific or ubiquitous expression of several AQPs in different tissues of jujube. Interestingly, TIP3−1 was found to have fruit specific expression whereas most of the AQPs have a relatively low expression. Based on the present study and previous reports, TIP3s seems to have a significant role in seed desiccation processes. The findings presented here provide pivotal insights into the functions of extremophile specific AQPs, to better understand the role of AQPs and, subsequently, the stress tolerance mechanism in jujube.

A Survey of Barley PIP Aquaporin Ionic Conductance Reveals Ca2+-Sensitive HvPIP2;8 Na+ and K+ Conductance.

Some plasma membrane intrinsic protein (PIP) aquaporins can facilitate ion transport. Here we report that one of the 12 barley PIPs (PIP1 and PIP2) tested, HvPIP2;8, facilitated cation transport when expressed in Xenopus laevis oocytes. HvPIP2;8-associated ion currents were detected with Na+ and K+, but not Cs+, Rb+, or Li+, and was inhibited by Ba2+, Ca2+, and Cd2+ and to a lesser extent Mg2+, which also interacted with Ca2+. Currents were reduced in the presence of K+, Cs+, Rb+, or Li+ relative to Na+ alone. Five HvPIP1 isoforms co-expressed with HvPIP2;8 inhibited the ion conductance relative to HvPIP2;8 alone but HvPIP1;3 and HvPIP1;4 with HvPIP2;8 maintained the ion conductance at a lower level. HvPIP2;8 water permeability was similar to that of a C-terminal phosphorylation mimic mutant HvPIP2;8 S285D, but HvPIP2;8 S285D showed a negative linear correlation between water permeability and ion conductance that was modified by a kinase inhibitor treatment. HvPIP2;8 transcript abundance increased in barley shoot tissues following salt treatments in a salt-tolerant cultivar Haruna-Nijo, but not in salt-sensitive I743. There is potential for HvPIP2;8 to be involved in barley salt-stress responses, and HvPIP2;8 could facilitate both water and Na+/K+ transport activity, depending on the phosphorylation status.

Genome-Wide Identification, Evolution, and Expression Profile of Aquaporin Genes in Coffea canephora in Response to Water Deficit

Aquaporins (APQs) belong to the major intrinsic protein superfamily and play a key role in the transport of water and other solutes across cell membranes. Coffea canephora is an evergreen shrub used for making instant coffees. Genome analysis of C. canephora identified 33 putative aquaporin genes assigned to five subfamilies including seven plasma membrane intrinsic proteins (PIP), 9 tonoplast intrinsic proteins (TIP), 11 NOD26-like intrinsic proteins (NIP), 3 small basic intrinsic proteins (SIP), and 3 X intrinsic proteins (XIP). Generally, the AQPs gene structure was conserved within each subfamily, with exon numbers ranging from one to five. The prediction of the aromatic/arginine selectivity filter (ar/R) and Froger’s positions indicated a noticeable difference in substrate specificity between subfamilies. Synteny analysis revealed high conservation of aquaporin genes in coffee. In silico expression analysis of the CcAQPs genes indicated that they were differentially expressed in various tissues. Members of CcPIPs and CcTIPs subfamilies were validated by real-time quantitative analysis in leaves of two genotypes of C. canephora with contrasting responses to water deficit (clone 14: drought-tolerant and clone 109A: drought-susceptible). Under severe water deficit, the relative expression of isoforms of both genes decreased in clone 14 compared with that under the irrigated condition, while clone 109A showed comparatively higher mRNA levels, with the exception of CcPIP1;2 in the stress condition. This study was the first to characterize and validate aquaporin genes in C. canephora in response to water deficit, and the findings may provide insights for biotechnological approaches to increase tolerance to drought.

Role of Protein-Water Interface in the Stacking Interactions of Granum Thylakoid Membranes-As Revealed by the Effects of Hofmeister Salts.

The thylakoid membranes of vascular plants are differentiated into stacked granum and unstacked stroma regions. The formation of grana is triggered by the macrodomain formation of photosystem II and light-harvesting complex II (PSII-LHCII) and thus their lateral segregation from the photosystem I—light-harvesting complex I (PSI-LHCI) super-complexes and the ATP-synthase; which is then stabilized by stacking interactions of the adjacent PSII-LHCII enriched regions of the thylakoid membranes. The self-assembly and dynamics of this highly organized membrane system and the nature of forces acting between the PSII-LHCII macrodomains are not well understood. By using circular dichroism (CD) spectroscopy, small-angle neutron scattering (SANS) and transmission electron microscopy (TEM), we investigated the effects of Hofmeister salts on the organization of pigment-protein complexes and on the ultrastructure of thylakoid membranes. We found that the kosmotropic agent (NH4)2SO4 and the Hofmeister-neutral NaCl, up to 2 M concentrations, hardly affected the macro-organization of the protein complexes and the membrane ultrastructure. In contrast, chaotropic salts, NaClO4, and NaSCN destroyed the mesoscopic structures, the multilamellar organization of the thylakoid membranes and the chiral macrodomains of the protein complexes but without noticeably affecting the short-range, pigment-pigment excitonic interactions. Comparison of the concentration- and time-dependences of SANS, TEM and CD parameters revealed the main steps of the disassembly of grana in the presence of chaotropes. It begins with a rapid diminishment of the long-range periodic order of the grana membranes, apparently due to an increased stacking disorder of the thylakoid membranes, as reflected by SANS experiments. SANS measurements also allowed discrimination between the cationic and anionic effects—in stacking and disorder, respectively. This step is followed by a somewhat slower disorganization of the TEM ultrastructure, due to the gradual loss of stacked membrane pairs. Occurring last is the stepwise decrease and disappearance of the long-range chiral order of the protein complexes, the rate of which was faster in LHCII-deficient membranes. These data are interpreted in terms of a theory, from our laboratory, according to which Hofmeister salts primarily affect the hydrophylic-hydrophobic interactions of proteins, and the stroma-exposed regions of the intrinsic membrane proteins, in particular—pointing to the role of protein-water interface in the stacking interactions of granum thylakoid membranes.