scholarly journals Mutation of Aquaporin Gene PIP2;5 Postpones Pollen Hydration in Arabidopsis

2018 ◽  
Author(s):  
Tan Su Jian ◽  
Juan Wang ◽  
Na Sui ◽  
Xian Sheng Zhang ◽  
Fang Wang
Keyword(s):  
Pollen Tube ◽  
Water Movement ◽  
Water Channel ◽  
Channel Activity ◽  
Wild Type ◽  
Pollen Hydration ◽  
Cell Plasma ◽  
Aquaporin Gene ◽  
Dna Insertion ◽  
Germination And Growth

In flowering plants with dry stigmas, pollen hydration involves water movement, which may be facilitated by aquaporins. To explore the possibility underlying this biological process, we identified and characterized a mutant with a T-DNA insertion in PIP2;5, which encodes an aquaporin with water channel activity in the PIP2 subfamily. We monitored the pollination process (pollen hydration, germination, and pollen tube growth) of wild type pollen on stigmas of the mutant and wild type. Pollen hydration was postponed on the stigmas of the mutant, compared with that on wild type stigmas. However, pollen tube germination and growth was unaffected in the mutant. The PIP2;5 protein was located in the cell plasma membrane and was preferentially expressed in the stigma. Based on our results, we concluded that PIP2;5 might play an important role in water movement during pollen hydration.

scholarly journals In Vivo Functional Assay of a Recombinant Aquaporin in Pichia pastoris

2006 ◽  
Vol 72 (2) ◽  
pp. 1507-1514 ◽  
Author(s):  
Mark J. Daniels ◽  
Malcolm R. Wood ◽  
Mark Yeager
Keyword(s):  
Pichia Pastoris ◽  
Linear Relationship ◽  
Osmotic Shock ◽  
Water Channel ◽  
Yeast Cells ◽  
Mercury Chloride ◽  
Functional Assay ◽  
Channel Activity ◽  
Wild Type

ABSTRACT The water channel protein PvTIP3;1 (α-TIP) is a member of the major intrinsic protein (MIP) membrane channel family. We overexpressed this eukaryotic aquaporin in the methylotrophic yeast Pichia pastoris, and immunogold labeling of cellular cryosections showed that the protein accumulated in the plasma membrane, as well as vacuolar and other intracellular membranes. We then developed an in vivo functional assay for water channel activity that measures the change in optical absorbance of spheroplasts following an osmotic shock. Spheroplasts of wild-type P. pastoris displayed a linear relationship between absorbance and osmotic shock level. However, spheroplasts of P. pastoris expressing PvTIP3;1 showed a break in this linear relationship corresponding to hypo-osmotically induced lysis. It is the difference between control and transformed spheroplasts under conditions of hypo-osmotic shock that forms the basis of our aquaporin activity assay. The aquaporin inhibitor mercury chloride blocked water channel activity but had no effect on wild-type yeast. Osmotically shocked yeast cells were affected only slightly by expression of the Escherichia coli glycerol channel GlpF, which belongs to the MIP family but is a weak water channel. The important role that aquaporins play in human physiology has led to a growing interest in their potential as drug targets for treatment of hypertension and congestive heart failure, as well as other fluid overload states. The simplicity of this assay that is specific for water channel activity should enable rapid screening for compounds that modulate water channel activity.

scholarly journals Two subgroups of receptor-like kinases promote early compatible pollen responses in the Arabidopsis thaliana pistil

2020 ◽  
Author(s):  
Hyun Kyung Lee ◽  
Daphne R Goring
Keyword(s):  
Arabidopsis Thaliana ◽  
Pollen Tube ◽  
Reproductive Tract ◽  
Cell Communication ◽  
Female Reproductive Tract ◽  
Peptide Ligands ◽  
Wild Type ◽  
Pollen Hydration ◽  
Early Stages ◽  
Compatible Pollen

Abstract In flowering plants, cell–cell communication between the compatible pollen grain/growing pollen tube and the pistil is an essential component for successful sexual reproduction. In Arabidopsis thaliana, the later stages of this dialogue are mediated by several peptide ligands and receptors that guide pollen tubes to the ovules for the release of sperm cells. Despite a detailed understanding of these processes, a key gap remains regarding the nature of the regulators that function at the earlier stages which are essential steps leading to fertilization. Here, we report on new functions for A. thaliana Receptor-Like Kinase (RLK) genes belonging to the LRR-II and LRR-VIII-2 RLK subgroups in the female reproductive tract to regulate compatible pollen hydration and the early stages of pollen tube growth. Mutant pistils for the A. thaliana RKF1 gene cluster were observed to support reduced wild-type pollen hydration and, when combined with the SERK1 and SERK3/BAK1 mutations, reduced pollen tube travel distances occurred. As these mutant pistils displayed a wild-type morphology, we propose that the observed altered compatible pollen responses result from an impaired pollen–pistil dialogue at these early stages.

scholarly journals BMP signaling alters aquaporin-4 expression in the mouse cerebral cortex

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kazuya Morita ◽  
Naoyuki Matsumoto ◽  
Kengo Saito ◽  
Toshihide Hamabe-Horiike ◽  
Keishi Mizuguchi ◽  
...  
Keyword(s):  
Cerebral Cortex ◽  
Molecular Mechanisms ◽  
Water Movement ◽  
Water Channel ◽  
Bmp Signaling ◽  
Aquaporin 4 ◽  
Mammalian Brain ◽  
Physiological Conditions ◽  
Pathological Conditions ◽  
Mouse Cerebral Cortex

AbstractAquaporin-4 (AQP4) is a predominant water channel expressed in astrocytes in the mammalian brain. AQP4 is crucial for the regulation of homeostatic water movement across the blood–brain barrier (BBB). Although the molecular mechanisms regulating AQP4 levels in the cerebral cortex under pathological conditions have been intensively investigated, those under normal physiological conditions are not fully understood. Here we demonstrate that AQP4 is selectively expressed in astrocytes in the mouse cerebral cortex during development. BMP signaling was preferentially activated in AQP4-positive astrocytes. Furthermore, activation of BMP signaling by in utero electroporation markedly increased AQP4 levels in the cerebral cortex, and inhibition of BMP signaling strongly suppressed them. These results indicate that BMP signaling alters AQP4 levels in the mouse cerebral cortex during development.

Mutation of a single amino acid converts the human water channel aquaporin 5 into an anion channel

2013 ◽  
Vol 305 (6) ◽  
pp. C663-C672 ◽  
Author(s):  
Xue Qin ◽  
Walter F. Boron
Keyword(s):  
Water Channel ◽  
Anion Channel ◽  
Single Amino Acid ◽  
Side Chain ◽  
Wild Type ◽  
Aquaporin 5 ◽  
Anion Conductance ◽  
Electrode Voltage ◽  
Microelectrode Measurements ◽  
Positively Charged

Aquaporin 6 (AQP6) is unique among mammalian AQPs in being an anion channel with negligible water permeability. However, the point mutation Asn60Gly converts AQP6 from an anion channel into a water channel. In the present study of human AQP5, we mutated Leu51 (corresponding to residue 61 in AQP6), the side chain of which faces the central pore. We evaluated function in Xenopus oocytes by two-electrode voltage clamp, video measurements of osmotic H2O permeability ( Pf), microelectrode measurements of surface pH (pHS) to assess CO2 permeability, and surface biotinylation. We found that AQP5-L51R does not exhibit the H2O or CO2 permeability of the wild-type protein but instead has a novel p-chloromercuribenzene sulfonate (pCMBS)-sensitive current. The double mutant AQP5-L51R/C182S renders the conductance insensitive to pCMBS, demonstrating that the current is intrinsic to AQP5. AQP5-L51R has the anion permeability sequence I− > NO3− ≅ NO2− > Br− > Cl− > HCO3− > gluconate. Of the other L51 mutants, L51T (polar uncharged) and L51V (nonpolar) retain H2O and CO2 permeability and do not exhibit anion conductance. L51D and L51E (negatively charged) have no H2O or CO2 permeability. L51K (positively charged) has an intermediate H2O and CO2 permeability and anion conductance. L51H is unusual in having a relatively low CO2 permeability and anion conductance, but a moderate Pf. Thus, positively charged mutations of L51 can convert AQP5 from a H2O/CO2 channel into an anion channel. However, the paradoxical effect of L51H is consistent with the hypothesis that CO2, in part, takes a pathway different from H2O through AQP5.

scholarly journals The Na+/H+ exchanger isoform 3 is required for active paracellular and transcellular Ca2+ transport across murine cecum

2013 ◽  
Vol 305 (4) ◽  
pp. G303-G313 ◽  
Author(s):  
Juraj Rievaj ◽  
Wanling Pan ◽  
Emmanuelle Cordat ◽  
R. Todd Alexander
Keyword(s):  
Water Movement ◽  
Water Flux ◽  
Wild Type Mouse ◽  
Wild Type ◽  
Rt Pcr ◽  
Ussing Chambers ◽  
Voltage Clamps ◽  
Paracellular Absorption ◽  
Mouse Intestine

Intestinal calcium (Ca2+) absorption occurs via paracellular and transcellular pathways. Although the transcellular route has been extensively studied, mechanisms mediating paracellular absorption are largely unexplored. Unlike passive diffusion, secondarily active paracellular Ca2+ uptake occurs against an electrochemical gradient with water flux providing the driving force. Water movement is dictated by concentration differences that are largely determined by Na+ fluxes. Consequently, we hypothesized that Na+ absorption mediates Ca2+ flux. NHE3 is central to intestinal Na+ absorption. NHE3 knockout mice (NHE3−/−) display impaired intestinal Na+, water, and Ca2+ absorption. However, the mechanism mediating this latter abnormality is not clear. To investigate this, we used Ussing chambers to measure net Ca2+ absorption across different segments of wild-type mouse intestine. The cecum was the only segment with net Ca2+ absorption. Quantitative RT-PCR measurements revealed cecal expression of all genes implicated in intestinal Ca2+ absorption, including NHE3. We therefore employed this segment for further studies. Inhibition of NHE3 with 100 μM 5-( N-ethyl- N-isopropyl) amiloride decreased luminal-to-serosal and increased serosal-to-luminal Ca2+ flux. NHE3−/− mice had a >60% decrease in luminal-to-serosal Ca2+ flux. Ussing chambers experiments under altered voltage clamps (−25, 0, +25 mV) showed decreased transcellular and secondarily active paracellular Ca2+ absorption in NHE3−/− mice relative to wild-type animals. Consistent with this, cecal Trpv6 expression was diminished in NHE3−/− mice. Together these results implicate NHE3 in intestinal Ca2+ absorption and support the theory that this is, at least partially, due to the role of NHE3 in Na+ and water absorption.

scholarly journals Two groups of Arabidopsis receptor kinases preferentially regulate the growth of intraspecies pollen tubes in the female reproductive tract

2020 ◽  
Author(s):  
Hyun Kyung Lee ◽  
Daphne R. Goring
Keyword(s):  
Pollen Tube ◽  
Reproductive Tract ◽  
Cell Communication ◽  
Pollen Grains ◽  
Pollen Tubes ◽  
Female Reproductive Tract ◽  
Wild Type ◽  
Knockout Mutant ◽  
Receptor Kinases ◽  
Compatible Pollen

SummaryIn flowering plants, continuous cell-cell communication between the compatible male pollen grain/growing pollen tube and the female pistil is required for successful sexual reproduction. In Arabidopsis thaliana, the later stages of this dialogue are mediated by several peptide ligands and receptor kinases that guide pollen tubes to the ovules for the release of sperm cells. Despite a detailed understanding of these processes, a key gap remains on the nature of the regulators that function at the earlier stages. Here, we report on two groups of A. thaliana receptor kinases, the LRR-VIII-2 RK subclass and the SERKs, that function in the female reproductive tract to regulate the compatible pollen grains and early pollen tube growth, both essential steps for the downstream processes leading to fertilization. Multiple A. thaliana LRR-VIII-2 RK and SERK knockout mutant combinations were created, and several phenotypes were observed such as reduced wild-type pollen hydration and reduced pollen tube travel distances. As these mutant pistils displayed a wild-type morphology, the observed altered responses of the wild-type pollen are proposed to result from the loss of these receptor kinases leading to an impaired pollen-pistil dialogue at these early stages. Furthermore, using pollen from related Brassicaceae species, we also discovered that these receptor kinases are required in the female reproductive tract to establish a reproductive barrier to interspecies pollen. Thus, we propose that the LRR-VIII-2 RKs and the SERKs play a dual role in the preferential selection and promotion of intraspecies pollen over interspecies pollen.

scholarly journals Light Gradient-Based Screening of Arabidopsis thaliana on a 384-Well Type Plant Array Chip

Micromachines ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 191
Author(s):  
Youn-Hee Park ◽  
Je-Kyun Park
Keyword(s):  
Arabidopsis Thaliana ◽  
White Light ◽  
Solid Medium ◽  
Red Light ◽  
Phytochrome B ◽  
Wild Type ◽  
Light Gradient ◽  
Gradient Based ◽  
Light Effects ◽  
Germination And Growth

Arabidopsis thaliana (Arabidopsis), as a model for plant research, is widely used for various aspects of plant science. To provide a more sophisticated and microscopic environment for the germination and growth of Arabidopsis, we report a 384-well type plant array chip in which each Arabidopsis seed is independently seeded in a solid medium. The plant array chip is made of a poly(methyl methacrylate) (PMMA) acrylic material and is assembled with a home-made light gradient module to investigate the light effects that significantly affect the germination and growth of Arabidopsis. The light gradient module was used to observe the growth pattern of seedlings according to the intensity of the white light and to efficiently screen for the influence of the white light. To investigate the response to red light (600 nm), which stimulates seed germination, the light gradient module was also applied to the germination test. As a result, the germination results showed that the plant array chip can be used to simultaneously screen wild type seeds and phytochrome B mutant seeds on a single array chip according to the eight red light intensities.

scholarly journals Carbon monoxide-releasing molecules inhibit the cystic fibrosis transmembrane conductance regulator Cl− channel

2020 ◽  
Vol 319 (6) ◽  
pp. L997-L1009
Author(s):  
Mayuree Rodrat ◽  
Walailak Jantarajit ◽  
Demi R. S. Ng ◽  
Bartholomew S. J. Harvey ◽  
Jia Liu ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Carbon Monoxide ◽  
Current Flow ◽  
Open Channels ◽  
Transmembrane Conductance Regulator ◽  
Channel Activity ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Flow Through ◽  
Cystic Fibrosis Transmembrane

The gasotransmitter carbon monoxide (CO) regulates fluid and electrolyte movements across epithelial tissues. However, its action on anion channels is incompletely understood. Here, we investigate the direct action of CO on the cystic fibrosis transmembrane conductance regulator (CFTR) by applying CO-releasing molecules (CO-RMs) to the intracellular side of excised inside-out membrane patches from cells heterologously expressing wild-type human CFTR. Addition of increasing concentrations of tricarbonyldichlororuthenium(II) dimer (CORM-2) (1–300 μM) inhibited CFTR channel activity, whereas the control RuCl3 (100 μM) was without effect. CORM-2 predominantly inhibited CFTR by decreasing the frequency of channel openings and, hence, open probability ( Po). But, it also reduced current flow through open channels with very fast kinetics, particularly at elevated concentrations. By contrast, the chemically distinct CO-releasing molecule CORM-3 inhibited CFTR by decreasing Po without altering current flow through open channels. Neither depolarizing the membrane voltage nor raising the ATP concentration on the intracellular side of the membrane affected CFTR inhibition by CORM-2. Interestingly, CFTR inhibition by CORM-2, but not by CFTRinh-172, was prevented by prior enhancement of channel activity by the clinically approved CFTR potentiator ivacaftor. Similarly, when added after CORM-2, ivacaftor completely relieved CFTR inhibition. In conclusion, CORM-2 has complex effects on wild-type human CFTR consistent with allosteric inhibition and open-channel blockade. Inhibition of CFTR by CO-releasing molecules suggests that CO regulates CFTR activity and that the gasotransmitter has tissue-specific effects on epithelial ion transport. The action of ivacaftor on CFTR Cl− channels inhibited by CO potentially expands the drug’s clinical utility.

CsSWEET1a and CsSWEET17 Mediate Growth and Freezing Tolerance by Promoting Sugar Transport across the Plasma Membrane

2020 ◽  
Vol 61 (9) ◽  
pp. 1669-1682
Author(s):  
Lina Yao ◽  
Changqing Ding ◽  
Xinyuan Hao ◽  
Jianming Zeng ◽  
Yajun Yang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Freezing Tolerance ◽  
Sugar Transport ◽  
Bimolecular Fluorescence Complementation ◽  
Freezing Stress ◽  
Sugar Uptake ◽  
Wild Type ◽  
Alternatively Spliced ◽  
Germination And Growth ◽  
Two Hybrid

Abstract Sugars Will Eventually be Exported Transporters (SWEETs) are important in plant biological processes. Expression levels of CsSWEET1a and CsSWEET17 are induced by cold acclimation (CA) and cold stress in Camellia sinensis. Here, we found that CsSWEET17 was alternatively spliced, and its exclusion (Ex) transcript was associated with the CA process. Both plasma membrane-localized CsSWEET1a and CsSWEET17 transport hexoses, but cytoplasm-localized CsSWEET17-Ex does not. These results indicate that alternative splicing may be involved in regulating the function of SWEET transporters in response to low temperature in plants. The extra C-terminal of CsSWEET17, which is not found in the tonoplast fructose transporter AtSWEET17, did not affect its plasma membrane localization but promoted its sugar transport activities. The overexpression (OE) of CsSWEET1a and CsSWEET17 genes resulted in an increased sugar uptake in Arabidopsis, affecting plant germination and growth. The leaf and seed sizes of the CsSWEET17-OE lines were significantly larger than those of the wild type. Moreover, the OE of CsSWEET1a and CsSWEET17 significantly reduced the relative electrolyte leakage levels under freezing stress. Compared with the wild type, the expression of AtCWINV genes was suppressed in both CsSWEET1a-OE and CsSWEET17-OE lines, indicating the alteration in sugar contents in the cell walls of the OE lines. Furthermore, the interaction between CsSWEET1a and CsSWEET17 was confirmed using yeast two-hybrid and bimolecular fluorescence complementation assays. We showed that CsSWEET1a and CsSWEET17 form homo-/heterodimers in the plasma membrane and mediate the partitioning of sugars between the cytoplasm and the apoplast, thereby regulating plant growth and freezing tolerance.

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