Global characterization of OsPIP aquaporins reveals that the H_(2)O_(2)transporter OsPIP2;6 increases resistance to rice blast

Plasma membrane intrinsic proteins(PIPs)are conserved plant aquaporins that transport small molecules across the plasma membrane to trigger instant stress responses and maintain cellular homeostasis under biotic and abiotic stress.To elucidate their roles in plant immunity to pathogen attack,we characterized the expression patterns,subcellular localizations,and H_(2)O_(2)-transport ability of 11 OsPIPs in rice(Oryza sativa),and identified OsPIP2;6 as necessary for rice disease resistance.OsPIP2;6 resides on the plasma membrane and facilitates cytoplasmic import of the immune signaling molecule H_(2)O_(2).Knockout of OsPIP2;6 increases rice susceptibility to Magnaporthe oryzae,indicating a positive function in plant immunity.OsPIP2;6 interacts with OsPIP2;2,which has been reported to increase rice resistance to pathogens via H_(2)O_(2)transport.Our findings suggest that OsPIP2;6 cooperates with OsPIP2;2 as a defense signal transporter complex during plant–pathogen interaction.

Plant aquaporins:Their roles beyond water transport

Compared to other organisms,plants have evolved a greater number of aquaporins with diverse substrates and functions to adapt to ever-changing environmental and internal stimuli for growth and development.Although aquaporins were initially identified as channels that allow water molecules to cross biological membranes,progress has been made in identifying various novel permeable substrates.Many studies have characterized the versatile physiological and biophysical functions of plant aquaporins.Here,we review the recent reports that highlight aquaporin-facilitated regulation of major physiological processes and stress tolerance throughout plant life cycles as well as the potential prospects and possibilities of applying aquaporins to improve agricultural productivity,food quality,environmental protection,and ecological conservation.

Subcellular Localization of Aquaporins OsPIP2-6 in Rice

[Objective] This study was to explore the subcellular localization of aquaporins OsPIP2-6 in rice. [Method] A key rice aquaporins gene OsPIP2-6 was cloned and used for construction of a transient expression vector,which was then transformed into onion epidermis via particle bombardment for confocal microscopy analysis using YFP gene as a reporter gene. [Result] The results showed that rice aquaporins OsPIP2-6 was mainly located in the plasma membrane. [Conclusion] Our results provided theoretical basis for further understanding plant aquaporins.

Aquaglyceroporins but not orthodox aquaporins are involved in the cryotolerance of pig spermatozoa

Background:Aquaporins(AQPs)are a family of transmembrane water channels that includes orthodox AQPs,aquaglyceroporins(GLPs)and super AQPs.AQP3,AQP7,AQP9 and AQP11 have been identified in boar sperm,and they are crucial for sperm maturation and osmoregulation.Water exchange is an important event in cryopreservation,which is the most efficient method for long-term storage of sperm.However,the freezethaw process leads to sperm damage and a loss of fertilizing potential.Assuming that the quality of frozenthawed sperm partially depends on the regulation of osmolality variations during this process,AQPs might play a crucial role in boar semen freezability.In this context,the aim of this study was to unravel the functional relevance of the different groups of AQPs for boar sperm cryotolerance through three different inhibitors.Results:Inhibition of different groups of AQPs was found to have different effects on boar sperm cryotolerance.Whereas the use of 1,3-propanediol(PDO),an inhibitor of orthodox AQPs and GLPs,decreased total motility(P<0.05),it increased post-thaw sperm viability,lowered membrane lipid disorder and increased mitochondrial membrane potential(MMP)(P<0.05).When acetazolamide(AC)was used as an inhibitor of orthodox AQPs,the effects on post-thaw sperm quality were restricted to a mild increase in MMP in the presence of the intermediate concentration at 30 min post-thaw and an increase in superoxide levels(P<0.05).Finally,the addition of phloretin(PHL),a GLP inhibitor,had detrimental effects on post-thaw total and progressive sperm motilities,viability and lipid membrane disorder(P<0.05).Conclusions:The effects of the different inhibitors suggest that GLPs rather than orthodox AQPs are relevant for boar sperm freezability.Moreover,the positive effect of PDO on sperm quality suggests a cryoprotective role for this molecule.

Involvement of aquaporins in a mouse model of rotavirus diarrhea

Rotavirus diarrhea is a major worldwide cause of infantile gastroenteritis; however, the mechanism responsible for intestinal fluid loss remains unclear. Water transfer across the intestinal epithelial membrane seems to occur because of aquaporins(AQPs). Accumulating evidence indicates that alterations in AQPs may play an important role in pathogenesis. Here, we focus on changes in AQPs in a mouse model of rotavirus diarrhea. In the present study, 32 of 35 mice developed diarrhea and mild dehydration within 24 hours after infection with rotavirus strain SA11. Intestinal epithelial cells demonstrated cytoplasmic vacuolation, malaligned villi, and atrophy. AQP1 expression was significantly attenuated in the ileum and colon in comparison with controls; likewise, AQP4 and-8 protein expression were significantly decreased in the colon of rotavirus diarrhea-infected mice. In contrast, AQP3 protein expression was significantly increased in the colon of rotavirus-infected mice in comparison with controls. These results indicate that rotavirus diarrhea is associated with the downregulation of AQP1,-4, and-8 expression. Therefore, AQPs play an important role in rotavirus diarrhea.

Aquaporins:Their role in cholestatic liver disease

This review focuses on current knowledge on hepato-cyte aquaporins(AQPs)and their significance in bile formation and cholestasis.Canalicular bile secretion results from a combined interaction of several solute transporters and AQP water channels that facilitate water flow in response to the osmotic gradients created. During choleresis,hepatocytes rapidly increase their canalicular membrane water permeability by modulating the abundance of AQP8.The question was raised as to whether the opposite process,i.e.a decreased canalicular AQP8 expression would contribute to the development of cholestasis.Studies in several experimental models of cholestasis,such as extrahepatic obstructive cholestasis,estrogen-induced cholestasis, and sepsis-induced cholestasis demonstrated that the protein expression of hepatocyte AQP8 was impaired. In addition,biophysical studies in canalicular plasma membranes revealed decreased water permeability associated with AQP8 protein downregulation.The combined alteration in hepatocyte solute transporters and AQP8 would hamper the efficient coupling of osmotic gradients and canalicular water flow.Thus cholestasis may result from a mutual occurrence of impaired solute transport and decreased water permeability.

Auphen and dibutyryl cAMP suppress growth of hepatocellular carcinoma by regulating expression of aquaporins 3 and 9 in vivo

AIM: To investigate whether the regulation of aquaporin 3 (AQP3) and AQP9 induced by Auphen and dibutyryl cAMP (dbcAMP) inhibits hepatic tumorigenesis.METHODS: Expression of AQP3 and AQP9 was detected by Western blot, immunohistochemistry (IHC), and RT-PCR in HCC samples and paired non-cancerous liver tissue samples from 30 hepatocellular carcinoma (HCC) patients. A xenograft tumor model was used in vivo. Nine nude mice were divided into control, Auphen-treated, and dbcAMP-treated groups (n = 3 for each group). AQP3 and AQP9 protein expression after induction of xenograft tumors was detected by IHC and mRNA by RT-PCR analysis. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and histological evaluation were used to detect apoptosis of tumor cells, and the concentration of serum &#x003b1;-fetoprotein (AFP) was measured using RT-PCR and an ELISA kit.RESULTS: The volumes and weights of tumors decreased significantly in the Auphen- and dbcAMP-treated mice compared with the control mice (P &#x0003c; 0.01). The levels of AQP3 were significantly lower in the Auphen treatment group, and levels of AQP9 were significantly higher in thedbcAMP treatment mice than in the control mice (P &#x0003c; 0.01). The reduction of AQP3 by Auphen and increase of AQP9 by dbcAMP in nude mice suppressed tumor growth of HCC, which resulted in reduced AFP levels in serum and tissues, and apoptosis of tumor cells in the Auphen- and dbcAMP-treated mice, when compared with control mice (P &#x0003c; 0.01). Compared with para-carcinoma tissues, AQP3 expression increased in tumor tissues whereas the expression of AQP9 decreased. By correlating clinicopathological and expression levels, we demonstrated that the expression of AQP3 and AQP9 was correlated with clinical progression of HCC and disease outcomes.CONCLUSION: AQP3 increases in HCC while AQP9 decreases. Regulation of AQP3 and AQP9 expression by Auphen and dbcAMP inhibits the development and growth of HCC.

Two aquaporins,PIP1;1 and PIP2;1,mediate the uptake of neonicotinoid pesticides in plants

Neonicotinoids(NEOs),a large class of organic compounds,are a type of commonly used pesticide for crop protection.Their uptake and accumulation in plants are prerequisites for their intra-and intercellular move-ments,transformation,and function.Understanding the molecular mechanisms that underpin NEO uptake by plants is crucial for effective application,which remains elusive.Here,we demonstrate that NEOs enter plant cells primarily through the transmembrane symplastic pathway and accumulate mainly in the cytosol.Two plasma membrane intrinsic proteins discovered in Brassica rapa,BraPIP1;1 and BraPIP2;1,were found to encode aquaporins(AQPs)that are highly permeable to NEOs in different plant species and facilitate NEO subcellular diffusion and accumulation.Their conserved transport function was further demonstrated in Xenopus laevis oocyte and yeast assays.BraPIP1;1 and BraPIP2;1 gene knockouts and interaction as-says suggested that their proteins can form functional heterotetramers.Assessment of the potential of mean force indicated a negative correlation between NEO uptake and the energy barrier of BraPIP1;1 chan-nels.This study shows that AQPs transport organic compounds with greater osmolarity than previously thought,providing new insight into the molecular mechanisms of organic compound uptake and facilitating innovations in systemic pesticides.

Hetong decoction(和通汤)relieves loperamide-induced constipation in rats by regulating expression of aquaporins

OBJECTIVE:To investigate whether Hetong decoction(和通汤,HTT)alleviates constipation via regulating AQPs expression.METHODS:Constipation in rats was induced by loperamide,and rats were randomly assigned into model(saline),HHT-low(95 g/kg),HTT-medium(190 g/kg),HTT-high(380 g/kg)and positive control(mosapride)groups.Then the defecation function,the concentration of serum arginine vasopressin(AVP)and cyclic adenosine monophosphate(cAMP),and the expression of AQP3 and AQP8 in colon tissues were assessed.NCM460 colon cells with AQP3 and AQP8 knockdown or overexpression were exposed to serum from rats that received low or high dose of HTT,followed by detection of AQP3 and AQP8 expression.RESULTS:The model group showed lower fecal weight and water content,weaker intestinal transit,higher serum concentration of AVP and cAMP,increased proximal and distal AQP8 expression,increased proximal but decreased distal AQP3 expression.However,these trends were reversed in both the HTT group(low,medium and high dose)and the positive control group.In NCM460 cells,HTT dose-dependently stabilized AQP3 and AQP8 expression under AQP3/8 plasmid interference or overexpression.CONCLUSIONS:HTT relieves constipation in rats through regulating AQP3 and AQP8 expression.

Effects of electroacupuncture on cochlear morphology and expression of aquaporins in rats with AVP-induced endolymphatic hydrops

Objective: The objective of this study was to investigate the effects of EA on EH and the regulation of AQP2 and AQP7 protein expression in rats.Methods: Twenty-four rats were allocated randomly to four groups of blank group, EH group,EH+tolvaptan group and EH + EA group(n = 6 per group). EH rat model was established by intraperitoneally injection of arginine vasopressin(AVP). EA was administered at acupoints "Baihui(百会 GV 20)"and "Tinggong(听宫 SI 19)". Rats in the EH + tolvaptan group and EH+ EA group were treated with tolvaptan and EA, respectively, after EH establishment. Hematoxylin-eosin staining was used to measure the cochlear hydrops degree, and then the ratio of scala media(SM) area to SM + scala vestibuli(SV) area(R value) was calculated. Immunohistochemical method was used to observe AQP2/AQP7 protein expression in the rat cochlear lateral wall after treatment.Results: ①There was no endolymphatic hydrops in the blank group. Reissner' s membrane was extended markedly and bulged into SV in cochleae of the EH group and endolymphatic hydrops was noted. Statistical analysis revealed that R value in the EH group showed a significant increase compared with that in the blank group(0.42 ± 0.02 vs. 0.31 ± 0.05, P=0.000). The distension of Reissner's membrane was less obvious in the EH + tolvaptan group and EH + EA group when compared with the EH group. R value in the EH + tolvaptan group and the EH + EA group was significantly less than that in EH group(0.32±0.04 vs. 0.42 ± 0.02, =0.001;0.35 ± 0.05 vs. 0.42 ± 0.02, P=0.012). The degree of the hydrops in the EH + EA group was not different from that in the EH + tolvaptan group(0.35 ± 0.05 vs. 0.32 ±0.04,P= 1.000). ②The AQP2 protein expression in the rat cochlear lateral wall of EH group was significantly increased when compared with the blank group(12.74 ± 5.18 vs. 5.92 ± 1.52, P = 0.014). The AQP2 protein expression in the rat cochlear lateral wall of EH + tolvaptan group and EH + EA group were all lower than that of the EH group(6.52 ± 2.73 vs. 12.74 ± 5.18. P = 0.029;6.95 ± 3.10 vs. 12.74 ± 5.18, P = 0.047).The AQP2 protein expression in the rat cochlear lateral wall of EH + EA group was not different from that in the EH + tolvaptan group(6.95 ± 3.10 vs. 6.52 ± 2.73, P= 1.000).③The AQP7 protein expression in the rat cochlear lateral wall of EH group was significantly increased when compared with the blank group(30.32 ± 6.39 vs 16.64 ± 3.21, P=0.000). The AQP7 protein expression in the rat cochlear lateral wall of EH + tolvaptan group and EH + EA group were all lower than that of the EH group(18.32 ± 2.45 vs.30.32 ± 6.39, P= 0.001;19.54 ± 4.61 vs. 30.32 ± 6.39, P= 0.003). The AQP7 protein expression in the rat cochlear lateral wall of EH + EA group was not different from that in the EH + tolvaptan group(19.54 ±4.61 vs. 18.32 ± 2.45, P= 1.000).Conclusions: These results indicate that repeated EA stimulation exerted the same effects as tolvaptan application on AQPs levels and subsequent aquaretic effects. And dehydrating effect of EA on the inner ear might be associated with its down-regulation of both AQP2 and AQP7 protein expression, thereby provide a potential molecular mechanism involved in the treatment of Meniere's disease by EA.

The regulatory roles of aquaporins in the digestive system

Aquaporins(AQPs)are highly conserved small transmembrane proteins,which are responsible for the water transport across the cell membrane.AQPs are abundantly expressed in numerous types of cells such as epithelial and endothelial cells.The expression of AQP-1,-3,-4,-5,-8 and-9 were found in the digestive system,where these six AQP isoforms serve essen-tial roles including mediating the transmembrane water transport and regulating the secretion of gastrointestinal(GI)fluids,consequently facilitating the digestion and absorption of GI con-tents.In addition,the expression levels of AQPs are controlled by various factors,and AQPs can stimulate numerous signaling pathways;however,aberrant expression of AQPs in the GI tracts are associated with the initiation and development of numerous diseases.Thus,this re-view provides an overview of the expression and functions of AQPs in the digestive system.

The Correlation between Nutrition and Transport Mechanism under Abiotic Stress in Plants: A Comprehensive Review

Variations in the nutrients and water that plants require for metabolism,development,and the maintenance of cellular homeostasis are the main causes of abiotic stress in plants.It has,however,hardly ever been studied how these transporter proteins,such as aquaporin which is responsible for food and water intake in cell plasma mem-branes,interact with one another.This review aims to explore the interactions between nutrient transporters and aquaporins during water and nutrient uptake.It also investigates how symbiotic relationships influence the plant genome’s responses to regulatory processes such as photoperiodism,senescence,and nitrogenfixation.These responses are observed in reaction to various abiotic stresses.For instance,plasma membrane transporters are upregulated during macronutrient insufficiency,tonoplast transporters are overexpressed,and aquaporins are downregulated in micronutrient deficiency.Additionally,tolerant plants often exhibit increased expression of nutrient transporters and aquaporins in response to drought,salt,and cold temperatures.To better comprehend plant stress tolerance to abiotic challenges including starvation,K famine,salt,and freezing temperatures,both classes of nutrient and water transporters should be considered at the same time.

Research Progress on the Pathogenesis of Acute Lung Injury(ALI)

In this review,the databases searched were PubMed and Web of Science.It is believed that the main causes of acute lung injury(ALI)and acute respiratory distress syndrome(ARDS)are inflammatory response disorders,excessive oxidative stress,cell death,endoplasmic reticulum stress,coagulation dysfunction,and weakened aquaporin function.

Deferential responses of plant aquaporins(AQPs) to osmotic and salt stresses

With the support of the National Natural Science Foundation of China,Prof.Ye Qing’s laboratory at South China Botanical Garden,Chinese Academy of Sciences,reported differential regulatory mechanisms of AQPs in mediating plant water transport under osmotic or salt stresses,which was published in Plant,Cell and Environment(2014,doi:10.1111/pce.12319).

Single Mutations in the Transmembrane Domains of Maize Plasma Membrane Aquaporins Affect the Activity of Monomers within a Heterotetramer

Aquaporins are channels facilitating the diffusion of water and/or small uncharged solutes ecross biological membranes. They assemble as homotetramers but some of them also form heterotetramers, especially in plants. In Zea mays, aquaporins belonging to the plasma membrane intrinsic protein （PIP） subfamily are clustered into two groups, PIP1 and PIP2, which exhibit different water-channel activities when expressed in Xenopus oocytes. When PIP1 and PIP2 isoforms are co-expressed, they physically interact to modulate their subcellular localization and channel activity. Here, we demonstrated by affinity chromatography purification that, when co-expressed in Xenopus oocytes, the maize PIP1;2 and PIP2;5 isoforms assemble as homo- and heterodimers within heterotetramers. We built the 3D structure of such heterotetramers by comparative modeling on the basis of the spinach SoPIP2;1 X-ray structure and identified amino acid residues in the transmembrane domains which putatively interact at the interfaces between monomers. Their roles in the water-channel activity, subcellular localization, protein abundance, and physical interac- tion were investigated by mutagenesis. We highlighted single-residue substitutions that either inactivated PIP2;5 or activated PIP1;2 without affecting their interaction. Interestingly, the Phe220Ala mutation in the transmembrane domain 5 of PIP1 ;2 activated its water-channel activity and, at the same time, inactivated PIP2;5 within a heterotetramer. Altogether, these data contribute to a better understanding of the interaction mechanisms between PIP isoforms and the role of heterotetramerization on their water-channel activity.

Progress on the Application of Aquaporins in Chinese Medicine

Aquapodns are a group of membrane proteins, which are known as the passages of water molecules transforming through the biological membrane lipid bilayer and distributing in almost all of the organs and tissues of living creatures. Aquaporins play important roles in maintaining water balance and internal environment stability. As a new entry point, aquaporins are involved in the researches on water metabolism, physiological regulation and pathological essence in viscera-state more and more widely in recent years. The literature on traditional Chinese medical studies, which related to aquaporins and were published in the last decade, was reviewed and the progress on application of aquaporin in Chinese medicine was summarized in this paper.

Ontogeny of the mammalian kidney:expression of aquaporins 1,2,3,and 4

Background:Determining the expression and functions of aquaporins(AQPs)in the adult kidney has generated important information about the roles of this protein family in the renal regulation of water homeostasis.However,limited information describes the expression of AQPs in fetal kidneys,and most reports on fetal renal AQPs originate from animal studies.Although there are the maturation and regulation of the renalconcentrating mechanism,the ways in which changes in the expression of AQPs contribute to the formation of urine during the perinatal period remain unclear.Data sources:This review summarizes current knowledge about the spatial and temporal expression patterns of AQP1,AQP2,AQP3,and AQP4 in the fetal and postnatal kidneys in different animal species and in human beings.Results:AQP1 and AQP2 expression can be detected earlier in gestation in human beings and sheep compared with mice and rats.AQP1 expression is detected earlier in the proximal tubules than the expression of AQP2,AQP3,and AQP4 in the collecting ducts.Conclusion:Further studies investigating the regulation of AQPs during kidney development may provide insights into normal water-handling mechanisms and the pathophysiology of fetal kidneys,which may determine new directions for the clinical treatment of kidney diseases.

Subcellular Redistribution of Root Aquaporins nduced by Hydrogen Peroxide

Aquaporins are water channel proteins that mediate the fine-tuning of cell membrane water permeability during development or in response to environmental stresses. The present work focuses on the oxidative stress-induced redistribution of plasma membrane intrinsic protein （PIP） aquaporins from the plasma membrane （PM） to intracellular membranes. This process was investigated in the Arabidopsis root. Su- crose density gradient centrifugation showed that exposure of roots to 0.5 mM H2O2 induces significant depletion in PM fractions of several abundant PIP homologs after 15 rain. Analyses by single-particle tracking and fluorescence correlative spectroscopy showed that, in the PM of epidermal cells, H2O2 treat- ment induces an increase in lateral motion and a reduction in the density of a fluorescently tagged form of the prototypal AtPIP2;1 isoform, respectively. Co-expression analyses of AtPIP2;1 with endomembrane markers revealed that H2O2 triggers AtPIP2;1 accumulation in the late endosomal compartments. Life- time analyses established that the high stability of PIPs was maintained under oxidative stress conditions, suggesting that H2O2 triggers a mechanism for intracellular sequestration of PM aquaporins without further degradation. In addition to information on cellular regulation of aquaporins, this study provides novel and complementary insights into the dynamic remodeling of plant internal membranes during oxida- tive stress responses.

Lights at night:does photobiomodulation improve sleep?

Sleep is a critical part of our daily routine.It impacts every organ and system of our body,from the brain to the heart and from cellular metabolism to immune function.A consistent daily schedule of quality of sleep makes a world of difference to our health and well-being.Despite its importance,so many individuals have trouble sleeping well.Poor quality sleep has such a detrimental impact on many aspects of our lives;it affects our thinking,learning,memory,and movements.Further,and most poignantly,poor quality sleep over time increases the risk of developing a serious medical condition,including neurodegenerative disease.In this review,we focus on a potentially new non-pharmacological treatment that improves the quality of sleep.This treatment,called photobiomodulation,involves the application of very specific wavelengths of light to body tissues.In animal models,these wavelengths,when applied at night,have been reported to stimulate the removal of fluid and toxic waste-products from the brain;that is,they improve the brain’s inbuilt house-keeping function.We suggest that transcranial nocturnal photobiomodulation,by improving brain function at night,will help improve the health and well-being of many individuals,by enhancing the quality of their sleep.

A cluster of mutagenesis revealed an osmotic regulatory role of the OsPIP1 genes in enhancing rice salt tolerance

Aquaporins play important regulatory roles in improving plant abiotic stress tolerance.To better understand whether the Os PIP1 genes collectively dominate the osmotic regulation in rice under salt stress,a cluster editing of the Os PIP1;1,Os PIP1;2 and Os PIP1;3 genes in rice was performed by CRISPR/Cas9 system.Sequencing showed that two mutants with Cas9-free,line 14 and line 18 were successfully edited.Briefly,line 14 deleted a single C base in both the Os PIP1;1 and Os PIP1;3 genes,and inserted a single T base in the Os PIP1;2 gene,respectively.While line 18 demonstrated an insertion of a single A base in the Os PIP1;1gene and a single T base in both the Os PIP1;2 and Os PIP1;3 genes,respectively.Multiplex editing of the Os PIP1 genes significantly inhibited photosynthetic rate and accumulation of compatible metabolites,but increased MDA contents and osmotic potentials in the mutants,thus delaying rice growth under salt stress.Functional loss of the Os PIP1 genes obviously suppressed the expressions of the Os PIP1,Os SOS1,Os CIPK24 and Os CBL4 genes,and increased the influxes of Na+and effluxes of K^(+)/H^(+)in the roots,thus accumulating more Na+in rice mutants under salt stress.This study suggests that the Os PIP1 genes are essential modulators collectively contributing to the enhancement of rice salt stress tolerance,and multiplex editing of the Os PIP1 genes provides insight into the osmotic regulation of the PIP genes.