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Summary We investigated the molecular basis and physiological implications of anion transport during pollen tube (PT) growth inArabidopsis thaliana(Col‐0).Patch‐clamp whole‐cell configuration analysis of pollen grain protoplasts revealed three subpopulations of anionic currents differentially regulated by cytoplasmic calcium ([Ca2+]cyt). We investigated the pollen‐expressed proteinsAtSLAH3,AtALMT12,AtTMEM16 andAtCCCas the putative anion transporters responsible for these currents.AtCCC‐GFPwas observed at the shank andAtSLAH3‐GFPat the tip and shank of thePTplasma membrane. Both are likely to carry the majority of anion current at negative potentials, as extracellular anionic fluxes measured at the tip ofPTs with an anion vibrating probe were significantly lower inslah3−/−andccc−/−mutants, but unaffected inalmt12−/−andtmem16−/−. We further characterised the effect ofpHandGABAby patch clamp. Strong regulation by extracellularpHwas observed in the wild‐type, but not intmem16−/−. Our results are compatible withAtTMEM16 functioning as an anion/H+cotransporter and therefore, as a putativepHsensor.GABApresence: (1) inhibited the overall currents, an effect that is abrogated in thealmt12−/−and (2) reduced the current inAtALMT12 transfectedCOS‐7 cells, strongly suggesting the direct interaction ofGABAwithAtALMT12.Our data show thatAtSLAH3 andAtCCCactivity is sufficient to explain the major component of extracellular anion fluxes, and unveils a possible regulatory system linkingPTgrowth modulation bypH,GABA, and [Ca2+]cytthrough anionic transporters.
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Cellular distribution of secretory pathway markers in the haploid synergid cells of Arabidopsis thaliana
Summary In flowering plants, cell–cell communication plays a key role in reproductive success, as both pollination and fertilization require pathways that regulate interactions between many different cell types. Some of the most critical of these interactions are those between the pollen tube (PT) and the embryo sac, which ensure the delivery of sperm cells required for double fertilization. Synergid cells function to attract thePTthrough secretion of small peptides and inPTreception via membrane‐bound proteins associated with the endomembrane system and the cell surface. While many synergid‐expressed components regulatingPTattraction and reception have been identified, few tools exist to study the localization of membrane‐bound proteins and the components of the endomembrane system in this cell type. In this study, we describe the localization and distribution of seven fluorescent markers that labelled components of the secretory pathway in synergid cells ofArabidopsis thaliana. These markers were used in co‐localization experiments to investigate the subcellular distribution of the twoPTreception componentsLORELEI, aGPI‐anchored surface protein, andNORTIA, aMILDEW RESISTANCE LOCUSO protein, both found within the endomembrane system of the synergid cell. These secretory markers are useful tools for both reproductive and cell biologists, enabling the analysis of membrane‐associated trafficking within a haploid cell actively involved in polar transport.
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- Award ID(s):
- 1733865
- PAR ID:
- 10054508
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- The Plant Journal
- Volume:
- 94
- Issue:
- 1
- ISSN:
- 0960-7412
- Page Range / eLocation ID:
- p. 192-202
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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