Membrane potential changes during pollen germination and tube growth

Breygina, Maria; Smirnova, A. V.; Матвеева, Н. П.; Yermakov, I. P.

doi:10.1134/s1990519x0906011x

Cited by 20 publications

(20 citation statements)

References 35 publications

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“…In particular, exogenously added H 2 O 2 caused plasma membrane hyperpolarization Podolyan et al 2019) in pollen tubes and protoplasts but the question remained whether this response appears in the presence of exudate or not. Here we demonstrate that hyperpolarization appears both in pollen tubes exhibiting normal MP gradient as reported earlier (Breygina et al 2010) and in pollen protoplasts treated with stigma exudate, and is abolished after catalase treatmentthus, hyperpolarization is caused by H 2 O 2 in the exudate and is a part of natural physiological crosstalk. The experimental system used can be considered as a useful model for assessment of membrane effects by flow cytometry.…”

Section: Discussionsupporting

confidence: 86%

“…MP of pollen protoplasts was assessed by staining with 1 µM DiBAC 4 (3) for 5 min (Breygina et al 2010) on Gallios flow cytometer (Beckman Coulter), analysis was performed with FlowJo software (TreeStar). Tinopal staining (1 mg/l) was used to distinguish between the population of protoplasts and cell debris, pollen walls and intact grains.…”

Section: Flow Cytometry Of Pollen Protoplastsmentioning

confidence: 99%

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ROS in tobacco stigma exudate affect pollen proteome and provoke membrane hyperpolarization

Barcikowska

Ekaterina

Eugeny

et al. 2020

Preprint

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ROS are known to be accumulated in stigmas of different species and can possibly perform different functions in plant reproduction. Here we confirm the assumption that they affect pollen by altering ion transport through the plasma membrane; as a more deferred effect, pollen proteome is modified. We detected ROS in stigma exudate, found hyperpolarization in exudatetreated growing pollen tubes and used flow cytometry of pollen protoplasts to compare the effects of fresh exudate and exogenous H 2 O 2 on pollen tube plasmalemma. Exudate causes plasmalemma hyperpolarization similar to the one provoked by H 2 O 2 , which is abolished by catalase treatment and ROS quencher MnTMPP. Inhibitory analysis indicates the participation of Ca 2+ -and K + -conducting channels in the observed hyperpolarization, linking obtained data with previous patch-clamp studies in vitro. For a deeper understanding of pollen response to ROS we analyzed proteome alterations in H 2 O 2 -treated pollen grains. We found 50 unique proteins and 20 differently accumulated proteins that are mainly involved in cell metabolism, energetics, protein synthesis and folding. Thus, pollen is getting ready for effective resource usage, construction of cellular components and rapid growth. Highlights The active substance in stigma exudate is H 2 O 2  H 2 O 2 causes hyperpolarization mediated by the activation of cation channels.  H 2 O 2 affects pollen proteome; we found 50 unique proteins.

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Section: Discussionsupporting

confidence: 86%

Section: Flow Cytometry Of Pollen Protoplastsmentioning

confidence: 99%

ROS in tobacco stigma exudate affect pollen proteome and provoke membrane hyperpolarization

Barcikowska

Ekaterina

Eugeny

et al. 2020

Preprint

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“…The existence of a voltage gradient along tip growing cells was discussed (Robinson, 1985;Holdaway-Clarke & Hepler, 2003;Geitmann & Palanivelu, 2007) on the basis of ion fluxes in distinct regions (Feij o et al, 1999;Zonia et al, 2002) and current recordings through growing PTs (Jaffe et al, 1974;Weisenseel et al, 1975). Attempts to measure membrane potential differences along growing PTs with sharp microelectrodes did not reveal such gradients (Robinson & Messerli, 2003) but have recently been reported by means of voltagesensitive dyes (Breygina et al, 2009).…”

Section: Correlation Between Tip-localized Anion-and Ca 2+homeostasismentioning

confidence: 99%

“…On the one hand, uptake of anions into the PTs serves the fast osmotically driven growth process; on the other hand, tip efflux of anions strongly correlates with PT growth rate (Zonia et al, 2001(Zonia et al, , 2002Gutermuth et al, 2013). Leakage of anions from pollen grains is essential to initiate pollen germination, while tube growth is inhibited when anion fluxes are disrupted pharmacologically (Zonia et al, 2002;Breygina et al, 2009Breygina et al, , 2010Breygina et al, , 2012Gutermuth et al, 2013). Apical anion fluxes are two to three orders of magnitude greater than the ones for Ca 2+ and H + , suggesting that they likely play an important physiological role in PT growth control as well (Holdaway-Clarke et al, 1997;Certal et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

Tip‐localized Ca²⁺‐permeable channels control pollen tube growth via kinase‐dependent R‐ and S‐type anion channel regulation

Gutermuth¹,

Herbell²,

Lassig

et al. 2018

New Phytologist

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Pollen tubes (PTs) are characterized by having tip-focused cytosolic calcium ion (Ca ) concentration ([Ca ] ) gradients, which are believed to control PT growth. However, the mechanisms by which the apical [Ca ] orchestrates PT growth are not well understood. Here, we aimed to identify these mechanisms by combining reverse genetics, cell biology, electrophysiology, and live-cell Ca and anion imaging. We triggered Ca -channel activation by applying hyperpolarizing voltage pulses and observed that the evoked [Ca ] increases were paralleled by high anion channel activity and a decrease in the cytosolic anion concentration at the PT tip. We confirmed a functional correlation between these patterns by showing that inhibition of Ca -permeable channels eliminated the [Ca ] increase, resulting in the abrogation of anion channel activity via Ca -dependent protein kinases (CPKs). Functional characterization of CPK and anion-channel mutants revealed a CPK2/20/6-dependent activation of SLAH3 and ALMT12/13/14 anion channels. The impaired growth phenotypes of anion channel and CPK mutants support the physiological significance of a kinase- and Ca -dependent pathway to control PT growth via anion channel activation. Other than unveiling this functional link, our membrane hyperpolarization method allows for unprecedented manipulation of the [Ca ] gradient or oscillations in the PT tips and opens an array of opportunities for channel screenings.

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“…A difference in electric polarization of the plasma membrane of growing pollen tubes has recently been reported (Breygina et al, 2009) using spectroscopic techniques and could explain the differential channel activity of voltage-dependent K + and Ca 2+ channels, reported to exist in the pollen tube plasma membrane (Fan et al, 2001;Mouline et al, 2002;Wang et al, 2004;Shang et al, 2005;Qu et al, 2007;Wu et al, 2007). This is a tantalizing hypothesis, because it would permit the generation of the biophysical conditions necessary for differential plasma membrane polarization domains, a mechanism we are currently trying to address.…”

Section: What Is the Role Of Anion Currents During Pollen Tube Growth?mentioning

confidence: 99%

Pollen Tube Growth Regulation by Free Anions Depends on the Interaction between the Anion Channel SLAH3 and Calcium-Dependent Protein Kinases CPK2 and CPK20

Gutermuth¹,

et al. 2013

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(J.A.F.).Apical growth in pollen tubes (PTs) is associated with the presence of tip-focused ion gradients and fluxes, implying polar localization or regulation of the underlying transporters. The molecular identity and regulation of anion transporters in PTs is unknown. Here we report a negative gradient of cytosolic anion concentration focused on the tip, in negative correlation with the cytosolic Ca 2+ concentration. We hypothesized that a possible link between these two ions is based on the presence of Ca 2+ -dependent protein kinases (CPKs). We characterized anion channels and CPK transcripts in PTs and analyzed their localization. Yellow fluorescent protein (YFP) tagging of a homolog of SLOW ANION CHANNEL-ASSOCIATED1 (SLAH3:YFP) was widespread along PTs, but, in accordance with the anion efflux, CPK2/CPK20/CPK17/CPK34:YFP fluorescence was strictly localized at the tip plasma membrane. Expression of SLAH3 with either CPK2 or CPK20 (but not CPK17/CPK34) in Xenopus laevis oocytes elicited S-type anion channel currents. Interaction of SLAH3 with CPK2/CPK20 (but not CPK17/CPK34) was confirmed by Förster-resonance energy transfer fluorescence lifetime microscopy in Arabidopsis thaliana mesophyll protoplasts and bimolecular fluorescence complementation in living PTs. Compared with wild-type PTs, slah3-1 and slah3-2 as well as cpk2-1 cpk20-2 PTs had reduced anion currents. Double mutant cpk2-1 cpk20-2 and slah3-1 PTs had reduced extracellular anion fluxes at the tip. Our studies provide evidence for a Ca 2+ -dependent CPK2/CPK20 regulation of the anion channel SLAH3 to regulate PT growth.

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Membrane potential changes during pollen germination and tube growth

Cited by 20 publications

References 35 publications

ROS in tobacco stigma exudate affect pollen proteome and provoke membrane hyperpolarization

ROS in tobacco stigma exudate affect pollen proteome and provoke membrane hyperpolarization

Tip‐localized Ca²⁺‐permeable channels control pollen tube growth via kinase‐dependent R‐ and S‐type anion channel regulation

Pollen Tube Growth Regulation by Free Anions Depends on the Interaction between the Anion Channel SLAH3 and Calcium-Dependent Protein Kinases CPK2 and CPK20

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Membrane potential changes during pollen germination and tube growth

Cited by 20 publications

References 35 publications

ROS in tobacco stigma exudate affect pollen proteome and provoke membrane hyperpolarization

ROS in tobacco stigma exudate affect pollen proteome and provoke membrane hyperpolarization

Tip‐localized Ca2+‐permeable channels control pollen tube growth via kinase‐dependent R‐ and S‐type anion channel regulation

Pollen Tube Growth Regulation by Free Anions Depends on the Interaction between the Anion Channel SLAH3 and Calcium-Dependent Protein Kinases CPK2 and CPK20

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Tip‐localized Ca²⁺‐permeable channels control pollen tube growth via kinase‐dependent R‐ and S‐type anion channel regulation