Detection of extracellular calcium gradients with a calcium-specific vibrating electrode.

Kühtreiber, Wiel M.; Jaffe, Lionel F.

doi:10.1083/jcb.110.5.1565

Cited by 237 publications

(187 citation statements)

References 20 publications

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“…Several lines of evidence suggest that PM Ca 21 channels may be prominently involved in the regulation of this Ca 21 gradient (Pierson et al, 1994(Pierson et al, , 1996Malhó et al, 1995). Ca 21 influx in pollen or pollen tubes has been investigated by using 45 Ca 21 (Jaffe et al, 1975), Ca 21 -selective vibrating probes (Kuhtreiber and Jaffe, 1990;Pierson et al, 1994;Holdaway-Clarke et al, 1997;Messerli et al, 1999;Franklin-Tong et al, 2002), and Mn 21 -quenching techniques . However, direct detection of Ca 21 channels in the PM of pollen or pollen tubes by using patch-clamp techniques has not been reported.…”

Section: Discussionmentioning

confidence: 99%

Ca2+-Permeable Channels in the Plasma Membrane of Arabidopsis Pollen Are Regulated by Actin Microfilaments

et al. 2004

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Cytosolic free Ca 21 and actin microfilaments play crucial roles in regulation of pollen germination and tube growth. The focus of this study is to test the hypothesis that Ca 21 channels, as well as channel-mediated Ca 21 influxes across the plasma membrane (PM) of pollen and pollen tubes, are regulated by actin microfilaments and that cytoplasmic Ca 21 in pollen and pollen tubes is consequently regulated. In vitro Arabidopsis (Arabidopsis thaliana) pollen germination and tube growth were significantly inhibited by Ca 21 channel blockers La 31 or Gd 31 and F-actin depolymerization regents. The inhibitory effect of cytochalasin D (CD) or cytochalasin B (CB) on pollen germination and tube growth was enhanced by increasing external Ca 21 . Ca 21 fluorescence imaging showed that addition of actin depolymerization reagents significantly increased cytoplasmic Ca 21 levels in pollen protoplasts and pollen tubes, and that cytoplasmic Ca 21 increase induced by CD or CB was abolished by addition of Ca 21 channel blockers. By using patch-clamp techniques, we identified the hyperpolarization-activated inward Ca 21 currents across the PM of Arabidopsis pollen protoplasts. The activity of Ca 21 -permeable channels was stimulated by CB or CD, but not by phalloidin. However, preincubation of the pollen protoplasts with phalloidin abolished the effects of CD or CB on the channel activity. The presented results demonstrate that the Ca 21 -permeable channels exist in Arabidopsis pollen and pollen tube PMs, and that dynamic actin microfilaments regulate Ca 21 channel activity and may consequently regulate cytoplasmic Ca 21 .The primary function of pollen and pollen tubes is to deliver sperms to egg apparatus for double fertilization that is required for sexual reproduction of flowering plants. Pollen germination and pollen tube growth are a continuous and highly polarized process characteristic of tip growth; thus pollen and pollen tubes provide an ideal model system for the study of cell polarity control and tip growth. It is well known that extracellular Ca 21 is required for pollen germination and tube growth (for review, see Steer and Steer, 1989;Taylor and Hepler, 1997;Malhó , 1998;Franklin-Tong, 1999), which indicates a possible involvement of Ca 21 influx in pollen germination and tube growth. Upon pollen hydration and germination, cytoplasmic Ca 21 concentration ([Ca 21 ] i ) at the germinal aperture where the pollen tube emerges increases to a higher level than other regions, and a tip-focused Ca 21 gradient is then established and sustained while a pollen tube grows forward (Rathore et al., 1991;Pierson et al., 1994;Feijó et al., 1995). Disruption or modification of the Ca 21 gradient inhibits pollen tube growth (Miller et al., 1992) or changes its growth direction (Malhó and Trewavas, 1996). The tip-focused Ca 21 gradient oscillates in the pollen tubes of lily (Lilium longiflorum) and other species (Holdaway-Clarke et al., 1997;Messerli and Robinson, 1997;Messerli et al., 2000). Ca 21 mobilization from intracellular Ca...

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Section: Discussionmentioning

confidence: 99%

Ca2+-Permeable Channels in the Plasma Membrane of Arabidopsis Pollen Are Regulated by Actin Microfilaments

et al. 2004

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“…The SIET system measures static ionic/molecular concentrations and concentration gradients using ion-selective microelectrodes (55). The concentration gradient was measured by moving the electrode repeatedly between two positions along a predefined excursion (5-30 m) at a fixed frequency in the range of 0.3-0.5 Hz.…”

Section: Measurement Of Reverse-mode Ncx Activity-full-lengthmentioning

confidence: 99%

A Na+/Ca2+ Exchanger-like Protein (AtNCL) Involved in Salt Stress in Arabidopsis

Wang

Wei

et al. 2012

Journal of Biological Chemistry

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“…Disrupting the Ca 2ϩ gradient or blocking Ca 2ϩ influx inhibited pollen tube growth, dissipated the cytoplasmic streaming associated with the pollen tube elongation and eliminated the clear zone found at the tip of actively elongating pollen (7,12,20). Extracellular Ca 2ϩ influx at the pollen tube tip was reported and this influx was correlated with the intracellular Ca 2ϩ gradient and polarized growth of the tip (12,(21)(22)(23). Ca 2ϩ influx at the apex is speculated to be due to stretch-activated Ca 2ϩ channels at the tip (12,22), while Ca 2ϩ -ATPase pumps are implicated in maintaining the Ca 2ϩ gradients in the cytosol (7,17).…”

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confidence: 96%

A Pollen-specific Novel Calmodulin-binding Protein with Tetratricopeptide Repeats

Safadi

Reddy

2000

Journal of Biological Chemistry

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Calcium is essential for pollen germination and pollen tube growth. A large body of information has established a link between elevation of cytosolic Ca 2؉ at the pollen tube tip and its growth. Since the action of Ca 2؉ is primarily mediated by Ca 2؉ -binding proteins such as calmodulin (CaM), identification of CaM-binding proteins in pollen should provide insights into the mechanisms by which Ca 2؉ regulates pollen germination and tube growth. In this study, a CaM-binding protein from maize pollen (maize pollen calmodulin-binding protein, MPCBP) was isolated in a protein-protein interactionbased screening using 35 S-labeled CaM as a probe. MPCBP has a molecular mass of about 72 kDa and contains three tetratricopeptide repeats (TPR) suggesting that it is a member of the TPR family of proteins. MPCBP protein shares a high sequence identity with two hypothetical TPR-containing proteins from Arabidopsis. Using gel overlay assays and CaM-Sepharose binding, we show that the bacterially expressed MPCBP binds to bovine CaM and three CaM isoforms from Arabidopsis in a Ca 2؉ -dependent manner. To map the CaMbinding domain several truncated versions of the MPCBP were expressed in bacteria and tested for their ability to bind CaM. Based on these studies, the CaMbinding domain was mapped to an 18-amino acid stretch between the first and second TPR regions. Gel and fluorescence shift assays performed with CaM and a CaMbinding synthetic peptide further confirmed MPCBP binding to CaM. Western, Northern, and reverse transcriptase-polymerase chain reaction analysis have shown that MPCBP expression is specific to pollen. MPCBP was detected in both soluble and microsomal proteins. Immunoblots showed the presence of MPCBP in mature and germinating pollen. Pollen-specific expression of MPCBP, its CaM-binding properties, and the presence of TPR motifs suggest a role for this protein in Ca 2؉ -regulated events during pollen germination and growth.

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Detection of extracellular calcium gradients with a calcium-specific vibrating electrode.

Cited by 237 publications

References 20 publications

Ca2+-Permeable Channels in the Plasma Membrane of Arabidopsis Pollen Are Regulated by Actin Microfilaments

Ca2+-Permeable Channels in the Plasma Membrane of Arabidopsis Pollen Are Regulated by Actin Microfilaments

A Na+/Ca2+ Exchanger-like Protein (AtNCL) Involved in Salt Stress in Arabidopsis

A Pollen-specific Novel Calmodulin-binding Protein with Tetratricopeptide Repeats

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