CRISPR/Cas9-Mediated Gene Editing of Vacuolar ATPase Subunit D Mediates Phytohormone Biosynthesis and Virus Resistance in Rice

Abstract Background: Vacuolar ATPases (v-ATPases) are proton pumps for proton translocation across membranes that utilize energy derived from ATP hydrolysis; Previous research revealed Osv-ATPases mediates phytohormes levels and resistance in rice. Osv-ATPase subunit d (Osv-ATPase d) is part of an integral, membrane-embedded V0 complex of V-ATPases complex, whether Osv-ATPase d involves in phytohormes biosynthesis and resistance in rice remains unknown.Finding: The knockout mutant line (line 5) of Osv-ATPase d was generated using the CRISPR/Cas9 system, mutation of Osv-ATPase d did not show any detrimental effect on plant growth or yield productivity. Transcriptomic results showed Osv-ATPase d probably involved in mediating the biosynthesis of plant hormones and resistance in rice. Mutation of Osv-ATPase d significantly increased JA and ABA biosynthesis than wild type. Compared to wild type, mutation of Osv-ATPase d increased the resistance against Southern rice black-streaked dwarf virus (SRBSDV), however, decreased the resistance against Rice stripe virus (RSV) in rice. Conclusion: Taken together, our data reveal the Osv-ATPase d mediates phytohormone biosynthesis and virus resistance in rice, which can be selected as a potential target for resistance breeding in rice.

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Structural organization of the V-ATPase and its implications for regulatory assembly and disassembly

Biochemical Society Transactions ◽

10.1042/bst0361027 ◽

2008 ◽

Vol 36 (5) ◽

pp. 1027-1031 ◽

Cited By ~ 22

Author(s):

Meikel Diepholz ◽

Michael Börsch ◽

Bettina Böttcher

Keyword(s):

Binding Sites ◽

Plasma Membranes ◽

Atp Hydrolysis ◽

Proton Translocation ◽

Nucleotide Binding Sites ◽

Disassembly Process ◽

Conformational Constraints ◽

Membrane Bound ◽

Vacuolar Atpases ◽

Hydrolysis Activity

V-ATPases (vacuolar ATPases) are membrane-bound multiprotein complexes that are localized in the endomembrane systems of eukaryotic cells and in the plasma membranes of some specialized cells. They couple ATP hydrolysis with the transport of protons across membranes. On nutrient shortage, V-ATPases disassemble into a membrane-embedded part (V0), which contains the proton translocation machinery, and an extrinsic part (V1), which carries the nucleotide-binding sites. Disassembly decouples ATP hydrolysis and proton translocation. Furthermore, the disassembled parts are inactive, leading to an efficient shutdown of ATP consumption. On restoring the nutrient levels, V1 and V0 reassemble and restore ATP-hydrolysis activity coupled with proton translocation. This reversible assembly/disassembly process has certain conformational constraints, which are best fulfilled by adopting a unique conformation before disassembly.

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Interaction of dibutyltin-3-hydroxyflavone bromide with the 16 kDa proteolipid indicates the disposition of proton translocation sites of the vacuolar ATPase

Biochemical Journal ◽

10.1042/bj3170425 ◽

1996 ◽

Vol 317 (2) ◽

pp. 425-431 ◽

Cited By ~ 6

Author(s):

Glen HUGHES ◽

Michael A. HARRISON ◽

Yong-In KIM ◽

David E. GRIFFITHS ◽

Malcolm E. FINBOW ◽

...

Keyword(s):

Atpase Activity ◽

Binding Sites ◽

Fluorescence Enhancement ◽

Atp Hydrolysis ◽

Dissociation Constants ◽

Proton Translocation ◽

Vacuolar Atpase ◽

Proton Pumping ◽

Wild Type ◽

Expression Studies

The organotin complex dibutyltin-3-hydroxyflavone bromide [Bu2Sn(of)Br] has been shown to bind to the 16 kDa proteolipid of Nephrops norvegicus, either in the form of the native protein or after heterologous expression in Saccharomyces and assembly into a hybrid vacuolar H+-ATPase. Titration of Bu2Sn(of)Br against the 16 kDa proteolipid results in a marked fluorescence enhancement, consistent with binding to a single affinity site on the protein. Vacuolar ATPase-dependent ATP hydrolysis was also inhibited by Bu2Sn(of)Br, with the inhibition constant correlating well with dissociation constants determined for binding of Bu2Sn(of)Br complex to the proteolipid. The fluorescence enhancement produced by interaction of probe with proteolipid can be back-titrated by dicyclohexylcarbodiimide (DCCD), which covalently modifies Glu140 on helix-4 of the polypeptide. Expression of a mutant proteolipid in which Glu140 was changed to a glycine resulted in assembly of a vacuolar ATPase which was inactive in proton pumping and which had reduced ATPase activity. Co-expression studies with this mutant and wild-type proteolipids suggest that proton pumping can only occur in a vacuolar ATPase containing exclusively wild-type proteolipid. The fluorescent enhancement or affinity of Bu2Sn(of)Br for the mutant proteolipid was not significantly altered, with the organotin complex having no effect on residual ATPase activity. Interaction of the probe with mutant proteolipid was unaffected by DCCD. These data suggest an overlap in the binding sites for organotin and DCCD, and have implications for the organization and structure of proton-translocating pathways in the vacuolar H+-ATPase.

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Role of Vma21p in Assembly and Transport of the Yeast Vacuolar ATPase

Molecular Biology of the Cell ◽

10.1091/mbc.e04-06-0514 ◽

2004 ◽

Vol 15 (11) ◽

pp. 5075-5091 ◽

Cited By ~ 59

Author(s):

Per Malkus ◽

Laurie A. Graham ◽

Tom H. Stevens ◽

Randy Schekman

Keyword(s):

Atp Hydrolysis ◽

Proton Translocation ◽

Wild Type ◽

Vitro Assay ◽

Accessory Factor ◽

Transport Vesicles ◽

Er Export

The Saccharomyces cerevisiae vacuolar H+-ATPase (V-ATPase) is a multisubunit complex composed of a peripheral membrane sector (V1) responsible for ATP hydrolysis and an integral membrane sector (V0) required for proton translocation. Biogenesis of V0 requires an endoplasmic reticulum (ER)-localized accessory factor, Vma21p. We found that in vma21Δ cells, the major proteolipid subunit of V0 failed to interact with the 100-kDa V0 subunit, Vph1p, indicating that Vma21p is necessary for V0 assembly. Immunoprecipitation of Vma21p from wild-type membranes resulted in coimmunoprecipitation of all five V0 subunits. Analysis of vmaΔ strains showed that binding of V0 subunits to Vma21p was mediated by the proteolipid subunit Vma11p. Although Vma21p/proteolipid interactions were independent of Vph1p, Vma21p/Vph1p association was dependent on all other V0 subunits, indicating that assembly of V0 occurs in a defined sequence, with Vph1p recruitment into a Vma21p/proteolipid/Vma6p complex representing the final step. An in vitro assay for ER export was used to demonstrate preferential packaging of the fully assembled Vma21p/proteolipid/Vma6p/Vph1p complex into COPII-coated transport vesicles. Pulse-chase experiments showed that the interaction between Vma21p and V0 was transient and that Vma21p/V0 dissociation was concomitant with V0/V1 assembly. Blocking ER export in vivo stabilized the interaction between Vma21p and V0 and abrogated assembly of V0/V1. Although a Vma21p mutant lacking an ER-retrieval signal remained associated with V0 in the vacuole, this interaction did not affect the assembly of vacuolar V0/V1 complexes. We conclude that Vma21p is not involved in regulating the interaction between V0 and V1 sectors, but that it has a crucial role in coordinating the assembly of V0 subunits and in escorting the assembled V0 complex into ER-derived transport vesicles.

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The contribution of PIP2-type aquaporins to photosynthetic response to increased vapour pressure deficit

Journal of Experimental Botany ◽

10.1093/jxb/erab187 ◽

2021 ◽

Author(s):

D Israel ◽

S Khan ◽

C R Warren ◽

J J Zwiazek ◽

T M Robson

Keyword(s):

Vapour Pressure Deficit ◽

Air Humidity ◽

Wild Type ◽

Knockout Mutant ◽

Evaporative Demand ◽

Co2 Transport ◽

Whole Plant ◽

Knockout Mutants ◽

Leaf Level ◽

Low Humidity

Abstract The roles of different plasma membrane aquaporins (PIPs) in leaf-level gas exchange of Arabidopsis thaliana were examined using knockout mutants. Since multiple Arabidopsis PIPs are implicated in CO2 transport across cell membranes, we focused on identifying the effects of the knockout mutations on photosynthesis, and whether they are mediated through the control of stomatal conductance of water vapour (gs), mesophyll conductance of CO2 (gm) or both. We grew Arabidopsis plants in low and high humidity environments and found that the contribution of PIPs to gs was larger under low air humidity when the evaporative demand was high, whereas any effect of lacking PIP function was minimal under higher humidity. The pip2;4 knockout mutant had 44% higher gs than the wild type plants under low humidity, which in turn resulted in an increased net photosynthetic rate (Anet). We also observed a 23% increase in whole-plant transpiration (E) for this knockout mutant. The lack of functional AtPIP2;5 did not affect gs or E, but resulted in homeostasis of gm despite changes of humidity, indicating a possible role in regulating CO2 membrane permeability. CO2 transport measurements in yeast expressing AtPIP2;5 confirmed that this aquaporin is indeed permeable to CO2.

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Cryo-EM structure and dynamics of the green-light absorbing proteorhodopsin

Nature Communications ◽

10.1038/s41467-021-24429-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Stephan Hirschi ◽

David Kalbermatter ◽

Zöhre Ucurum ◽

Thomas Lemmin ◽

Dimitrios Fotiadis

Keyword(s):

Proton Translocation ◽

Functional Characterization ◽

Green Light ◽

Proton Donor ◽

Molecular Organization ◽

Primary Proton ◽

Proton Pumps ◽

Model Protein ◽

Dynamics Simulations ◽

And Function

AbstractThe green-light absorbing proteorhodopsin (GPR) is the archetype of bacterial light-driven proton pumps. Here, we present the 2.9 Å cryo-EM structure of pentameric GPR, resolving important residues of the proton translocation pathway and the oligomerization interface. Superposition with the structure of a close GPR homolog and molecular dynamics simulations reveal conformational variations, which regulate the solvent access to the intra- and extracellular half channels harbouring the primary proton donor E109 and the proposed proton release group E143. We provide a mechanism for the structural rearrangements allowing hydration of the intracellular half channel, which are triggered by changing the protonation state of E109. Functional characterization of selected mutants demonstrates the importance of the molecular organization around E109 and E143 for GPR activity. Furthermore, we present evidence that helices involved in the stabilization of the protomer interfaces serve as scaffolds for facilitating the motion of the other helices. Combined with the more constrained dynamics of the pentamer compared to the monomer, these observations illustrate the previously demonstrated functional significance of GPR oligomerization. Overall, this work provides molecular insights into the structure, dynamics and function of the proteorhodopsin family that will benefit the large scientific community employing GPR as a model protein.

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Altered V-ATPase expression in renal intercalated cells isolated from B1 subunit-deficient mice by fluorescence-activated cell sorting

AJP Renal Physiology ◽

10.1152/ajprenal.00394.2012 ◽

2013 ◽

Vol 304 (5) ◽

pp. F522-F532 ◽

Cited By ~ 21

Author(s):

Luca Vedovelli ◽

John T. Rothermel ◽

Karin E. Finberg ◽

Carsten A. Wagner ◽

Anie Azroyan ◽

...

Keyword(s):

Cell Sorting ◽

Collecting Duct ◽

Egfp Expression ◽

Intercalated Cells ◽

Wild Type ◽

Western Blots ◽

Atpase Subunit ◽

Protein Levels ◽

Baseline Conditions ◽

Deficient Mice

Unlike human patients with mutations in the 56-kDa B1 subunit isoform of the vacuolar proton-pumping ATPase (V-ATPase), B1-deficient mice (Atp6v1b1−/−) do not develop metabolic acidosis under baseline conditions. This is due to the insertion of V-ATPases containing the alternative B2 subunit isoform into the apical membrane of renal medullary collecting duct intercalated cells (ICs). We previously reported that quantitative Western blots (WBs) from whole kidneys showed similar B2 protein levels in Atp6v1b1−/− and wild-type mice (Păunescu TG, Russo LM, Da Silva N, Kovacikova J, Mohebbi N, Van Hoek AN, McKee M, Wagner CA, Breton S, Brown D. Am J Physiol Renal Physiol 293: F1915–F1926, 2007). However, WBs from renal medulla (including outer and inner medulla) membrane and cytosol fractions reveal a decrease in the levels of the ubiquitous V-ATPase E1 subunit. To compare V-ATPase expression specifically in ICs from wild-type and Atp6v1b1−/− mice, we crossed mice in which EGFP expression is driven by the B1 subunit promoter (EGFP-B1+/+ mice) with Atp6v1b1−/− mice to generate novel EGFP-B1−/− mice. We isolated pure IC populations by fluorescence-assisted cell sorting from EGFP-B1+/+ and EGFP-B1−/− mice to compare their V-ATPase subunit protein levels. We report that V-ATPase A, E1, and H subunits are all significantly downregulated in EGFP-B1−/− mice, while the B2 protein level is considerably increased in these animals. We conclude that under baseline conditions B2 upregulation compensates for the lack of B1 and is sufficient to maintain basal acid-base homeostasis, even when other V-ATPase subunits are downregulated.

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Constitutive expression, not a particular primary sequence, is the important feature of the H3 replacement variant hv2 in Tetrahymena thermophila.

Molecular and Cellular Biology ◽

10.1128/mcb.17.11.6303 ◽

1997 ◽

Vol 17 (11) ◽

pp. 6303-6310 ◽

Cited By ~ 37

Author(s):

L Yu ◽

M A Gorovsky

Keyword(s):

Protein Sequence ◽

Tetrahymena Thermophila ◽

Histone H3 ◽

Constitutive Expression ◽

Histone Variants ◽

Wild Type ◽

Ciliated Protozoan ◽

Knockout Mutant ◽

Double Knockout ◽

Constitutive Synthesis

Although quantitatively minor replication-independent (replacement) histone variants have been found in a wide variety of organisms, their functions remain unknown. Like the H3.3 replacement variants of vertebrates, hv2, an H3 variant in the ciliated protozoan Tetrahymena thermophila, is synthesized and deposited in nuclei of nongrowing cells. Although hv2 is clearly an H3.3-like replacement variant by its expression, sequence analysis indicates that it evolved independently of the H3.3 variants of multicellular eukaryotes. This suggested that it is the constitutive synthesis, not the particular protein sequence, of these variants that is important in the function of H3 replacement variants. Here, we demonstrate that the gene (HHT3) encoding hv2 or either gene (HHT1 or HHT2) encoding the abundant major H3 can be completely knocked out in Tetrahymena. Surprisingly, when cells lacking hv2 are starved, a major histone H3 mRNA transcribed by the HHT2 gene, which is synthesized little, if at all, in wild-type nongrowing cells, is easily detectable. Both HHT2 and HHT3 knockout strains show no obvious defect during vegetative growth. In addition, a mutant with the double knockout of HHT1 and HHT3 is viable while the HHT2 HHT3 double-knockout mutant is not. These results argue strongly that cells require a constitutively expressed H3 gene but that the particular sequence being expressed is not critical.

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Conversion at large intergenic regions of mitochondrial DNA in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.10.4.1530-1537.1990 ◽

1990 ◽

Vol 10 (4) ◽

pp. 1530-1537

Author(s):

P J Skelly ◽

G D Clark-Walker

Keyword(s):

Saccharomyces Cerevisiae ◽

Mitochondrial Dna ◽

Rrna Gene ◽

Supporting Evidence ◽

Unexpected Result ◽

Wild Type ◽

Mitochondrial Dna Deletion ◽

Direct Role ◽

Atpase Subunit ◽

Intergenic Regions

Saccharomyces cerevisiae mitochondrial DNA deletion mutants have been used to examine whether base-biased intergenic regions of the genome influence mitochondrial biogenesis. One strain (delta 5.0) lacks a 5-kilobase (kb) segment extending from the proline tRNA gene to the small rRNA gene that includes ori1, while a second strain (delta 3.7) is missing a 3.7-kb region between the genes for ATPase subunit 6 and glutamic acid tRNA that encompasses ori7 plus ori2. Growth of these strains on both fermentable and nonfermentable substrates does not differ from growth of the wild-type strain, indicating that the deletable regions of the genome do not play a direct role in the expression of mitochondrial genes. Examination of whether the 5- or 3.7-kb regions influence mitochondrial DNA transmission was undertaken by crossing strains and examining mitochondrial genotypes in zygotic colonies. In a cross between strain delta 5.0, harboring three active ori elements (ori2, ori3, and ori5), and strain delta 3.7, containing only two active ori elements (ori3 and ori5), there is a preferential recovery of the genome containing two active ori elements (37% of progeny) over that containing three active elements (20%). This unexpected result, suggesting that active ori elements do not influence transmission of respiratory-competent genomes, is interpreted to reflect a preferential conversion of the delta 5.0 genome to the wild type (41% of progeny). Supporting evidence for conversion over biased transmission is shown by preferential recovery of a nonparental genome in the progeny of a heterozygous cross in which both parental molecules can be identified by size polymorphisms.

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Mutation of a single residue in the ba3 oxidase specifically impairs protonation of the pump site

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1422434112 ◽

2015 ◽

Vol 112 (11) ◽

pp. 3397-3402 ◽

Cited By ~ 16

Author(s):

Christoph von Ballmoos ◽

Nathalie Gonska ◽

Peter Lachmann ◽

Robert B. Gennis ◽

Pia Ädelroth ◽

...

Keyword(s):

Electron Transfer ◽

Time Constant ◽

Thermus Thermophilus ◽

Proton Translocation ◽

Proton Pumping ◽

Wild Type ◽

Structural Variant ◽

In The Wild ◽

Membrane Bound ◽

Proton Uptake

The ba3-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound protein complex that couples electron transfer to O2 to proton translocation across the membrane. To elucidate the mechanism of the redox-driven proton pumping, we investigated the kinetics of electron and proton transfer in a structural variant of the ba3 oxidase where a putative “pump site” was modified by replacement of Asp372 by Ile. In this structural variant, proton pumping was uncoupled from internal electron transfer and O2 reduction. The results from our studies show that proton uptake to the pump site (time constant ∼65 μs in the wild-type cytochrome c oxidase) was impaired in the Asp372Ile variant. Furthermore, a reaction step that in the wild-type cytochrome c oxidase is linked to simultaneous proton uptake and release with a time constant of ∼1.2 ms was slowed to ∼8.4 ms, and in Asp372Ile was only associated with proton uptake to the catalytic site. These data identify reaction steps that are associated with protonation and deprotonation of the pump site, and point to the area around Asp372 as the location of this site in the ba3 cytochrome c oxidase.

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