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Abstract The phytohormone ethylene has numerous effects on plant growth and development. Its immediate precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), is a non-proteinogenic amino acid produced by ACC SYNTHASE (ACS). ACC is often used to induce ethylene responses. Here, we demonstrate that ACC exhibits ethylene-independent signaling inArabidopsis thalianareproduction. By analyzing anacsoctuple mutant with reduced seed set, we find that ACC signaling in ovular sporophytic tissue is involved in pollen tube attraction, and promotes secretion of the pollen tube chemoattractant LURE1.2. ACC activates Ca2+-containing ion currents via GLUTAMATE RECEPTOR-LIKE (GLR) channels in root protoplasts. In COS-7 cells expressing mossPpGLR1, ACC induces the highest cytosolic Ca2+elevation compared to all twenty proteinogenic amino acids. In ovules, ACC stimulates transient Ca2+elevation, and Ca2+influx in octuple mutant ovules rescues LURE1.2 secretion. These findings uncover a novel ACC function and provide insights for unraveling new physiological implications of ACC in plants.
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Reduced Expression of TMEM16A Impairs Nitric Oxide-Dependent Cl− Transport in Retinal Amacrine Cells
Postsynaptic cytosolic Cl − concentration determines whether GABAergic and glycinergic synapses are inhibitory or excitatory. We have shown that nitric oxide (NO) initiates the release of Cl − from acidic internal stores into the cytosol of retinal amacrine cells (ACs) thereby elevating cytosolic Cl − . In addition, we found that cystic fibrosis transmembrane conductance regulator (CFTR) expression and Ca 2+ elevations are necessary for the transient effects of NO on cytosolic Cl − levels, but the mechanism remains to be elucidated. Here, we investigated the involvement of TMEM16A as a possible link between Ca 2+ elevations and cytosolic Cl − release. TMEM16A is a Ca 2+ -activated Cl − channel that is functionally coupled with CFTR in epithelia. Both proteins are also expressed in neurons. Based on this and its Ca 2+ dependence, we test the hypothesis that TMEM16A participates in the NO-dependent elevation in cytosolic Cl − in ACs. Chick retina ACs express TMEM16A as shown by Western blot analysis, single-cell PCR, and immunocytochemistry. Electrophysiology experiments demonstrate that TMEM16A functions in amacrine cells. Pharmacological inhibition of TMEM16A with T16inh-AO1 reduces the NO-dependent Cl − release as indicated by the diminished shift in the reversal potential of GABA A receptor-mediated currents. We confirmed the involvement of TMEM16A in the NO-dependent Cl − release using CRISPR/Cas9 knockdown of TMEM16A. Two different modalities targeting the gene for TMEM16A ( ANO1 ) were tested in retinal amacrine cells: an all-in-one plasmid vector and crRNA/tracrRNA/Cas9 ribonucleoprotein. The all-in-one CRISPR/Cas9 modality did not change the expression of TMEM16A protein and produced no change in the response to NO. However, TMEM16A-specific crRNA/tracrRNA/Cas9 ribonucleoprotein effectively reduces both TMEM16A protein levels and the NO-dependent shift in the reversal potential of GABA-gated currents. These results show that TMEM16A plays a role in the NO-dependent Cl − release from retinal ACs.
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- Award ID(s):
- 2129683
- PAR ID:
- 10420447
- Date Published:
- Journal Name:
- Frontiers in Cellular Neuroscience
- Volume:
- 16
- ISSN:
- 1662-5102
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3389/fncel.2022.937060
