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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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The Arabidopsis heterotrimeric G‐protein β subunit, AGB 1, is required for guard cell calcium sensing and calcium‐induced calcium release
<bold>Summary</bold> Cytosolic calcium concentration ([Ca2+]cyt) and heterotrimeric G‐proteins are universal eukaryotic signaling elements. In plant guard cells, extracellular calcium (Cao) is as strong a stimulus for stomatal closure as the phytohormone abscisic acid (ABA), but underlying mechanisms remain elusive. Here, we report that the sole Arabidopsis heterotrimeric Gβ subunit,AGB1, is required for four guard cell Caoresponses: induction of stomatal closure; inhibition of stomatal opening; [Ca2+]cytoscillation; and inositol 1,4,5‐trisphosphate (InsP3) production. Stomata in wild‐type Arabidopsis (Col) and in mutants of the canonical Gα subunit,GPA1, showed inhibition of stomatal opening and promotion of stomatal closure by Cao. By contrast, stomatal movements ofagb1mutants andagb1/gpa1double‐mutants, as well as those of theagg1agg2 Gγ double‐mutant, were insensitive to Cao. These behaviors contrast withABA‐regulated stomatal movements, which involveGPA1 andAGB1/AGG3 dimers, illustrating differential partitioning of G‐protein subunits among stimuli with similar ultimate impacts, which may facilitate stimulus‐specific encoding.AGB1knockouts retained reactive oxygen species andNOproduction, but lostYC3.6‐detected [Ca2+]cytoscillations in response to Cao, initiating only a single [Ca2+]cytspike. Experimentally imposed [Ca2+]cytoscillations restored stomatal closure inagb1. Yeast two‐hybrid and bimolecular complementation fluorescence experiments revealed thatAGB1 interacts with phospholipase Cs (PLCs), and Caoinduced InsP3 production in Col but not inagb1. In sum, G‐protein signaling viaAGB1/AGG1/AGG2 is essential for Cao‐regulation of stomatal apertures, and stomatal movements in response to Caoapparently require Ca2+‐induced Ca2+release that is likely dependent on Gβγ interaction withPLCs leading to InsP3 production.
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- PAR ID:
- 10460555
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- The Plant Journal
- Volume:
- 99
- Issue:
- 2
- ISSN:
- 0960-7412
- Page Range / eLocation ID:
- p. 231-244
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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