Cyt Oscillations Research Articles

AtFH5-labeled secretory vesicles-dependent calcium oscillation drives exocytosis and stepwise bulge during pollen germination

Pollen germination is an essential step for delivering sperm cells to the embryo sac for double fertilization inflowering plants. The cytosolic Ca2+ concentration ([Ca2+]cyt) and vesicle dynamics are critical for pollengermination, but their potential correlation in pollen grains is not fully understood. Here, we report that [Ca2+]cyt oscillates periodically at the prospective germination sites during pollen germination. The [Ca2+]cyt is mainly from extracellular Ca2+ ([Ca2+]ext) influx, which implicates the Ca2+-permeable ion channel cyclic nucleotide-gated channel 18 (CNGC18). The [Ca2+]cyt oscillations spatiotemporally correlate with the accumulation of secretory vesicles labeled by a formin protein AtFH5, and disruption of vesicle accumulation inhibits the [Ca2+]cyt oscillations. In turn, the [Ca2+]cyt oscillations promote exocytosis, which leads to stepwise cell extension during pollen germination. Together, these data provide a timeline of vesicle dynamics, calcium oscillation, and exocytosis during pollen germination and highlight the importance of the correlation of these events for pollen germination.

A novel calmodulin-interacting Domain of Unknown Function 506 protein represses root hair elongation in Arabidopsis.

Domain of Unknown Function 506 proteins are ubiquitous in plants. The phosphorus (P) stress‐inducible REPRESSOR OF EXCESSIVE ROOT HAIR GROWTH1 (AtRXR1) gene encodes the first characterized DUF506. AtRXR1 inhibits root hair elongation by interacting with RabD2c GTPase. However, functions of other P‐responsive DUF506 genes are still missing. Here, we selected two additional P‐inducible DUF506 genes for further investigation. The expression of both genes was induced by auxin. Under P‐stress, At3g07350 gene expressed ubiquitously in seedlings, whereas At1g62420 (AtRXR3) expression was strongest in roots. AtRXR3 overexpressors and knockouts had shorter and longer root hairs, respectively. A functional AtRXR3‐green fluorescent protein fusion localized to root epidermal cells. Chromatin immunoprecipitation and quantitative reverse‐transcriptase‐polymerase chain reaction revealed that AtRXR3 was transcriptionally activated by RSL4. Bimolecular fluorescence complementation and calmodulin (CaM)‐binding assays showed that AtRXR3 interacted with CaM in the presence of Ca2+. Moreover, cytosolic Ca2+ ([Ca2+]cyt) oscillations in root hairs of rxr3 mutants exhibited elevated frequencies and dampened amplitudes compared to those of wild type. Thus, AtRXR3 is another DUF506 protein that attenuates P‐limitation‐induced root hair growth through mechanisms that involve RSL4 and interaction with CaM to modulate tip‐focused [Ca2+]cyt oscillations.

A Ratiometric Calcium Reporter CGf Reveals Calcium Dynamics Both in the Single Cell and Whole Plant Levels Under Heat Stress.

Land plants evolved to quickly sense and adapt to temperature changes, such as hot days and cold nights. Given that calcium (Ca2+) signaling networks are implicated in most abiotic stress responses, heat-triggered changes in cytosolic Ca2+ were investigated in Arabidopsis leaves and pollen. Plants were engineered with a reporter called CGf, a ratiometric, genetically encoded Ca2+ reporter with an mCherry reference domain fused to an intensiometric Ca2+ reporter GCaMP6f. Relative changes in [Ca2+]cyt were estimated based on CGf’s apparent KD around 220 nM. The ratiometric output provided an opportunity to compare Ca2+ dynamics between different tissues, cell types, or subcellular locations. In leaves, CGf detected heat-triggered cytosolic Ca2+ signals, comprised of three different signatures showing similarly rapid rates of Ca2+ influx followed by differing rates of efflux (50% durations ranging from 5 to 19 min). These heat-triggered Ca2+ signals were approximately 1.5-fold greater in magnitude than blue light-triggered signals in the same leaves. In contrast, growing pollen tubes showed two different heat-triggered responses. Exposure to heat caused tip-focused steady growth [Ca2+]cyt oscillations to shift to a pattern characteristic of a growth arrest (22%), or an almost undetectable [Ca2+]cyt (78%). Together, these contrasting examples of heat-triggered Ca2+ responses in leaves and pollen highlight the diversity of Ca2+ signals in plants, inviting speculations about their differing kinetic features and biological functions.

A Dynamic Model of Cytosolic Calcium Concentration Oscillations in Mast Cells

In this paper, a dynamic model of cytosolic calcium concentration ([Ca2+]Cyt) oscillations is established for mast cells (MCs). This model includes the cytoplasm (Cyt), endoplasmic reticulum (ER), mitochondria (Mt), and functional region (μd), formed by the ER and Mt, also with Ca2+ channels in these cellular compartments. By this model, we calculate [Ca2+]Cyt oscillations that are driven by distinct mechanisms at varying kdeg (degradation coefficient of inositol 1,4,5-trisphosphate, IP3 and production coefficient of IP3), as well as at different distances between the ER and Mt (ER–Mt distance). The model predicts that (i) Mt and μd compartments can reduce the amplitude of [Ca2+]Cyt oscillations, and cause the ER to release less Ca2+ during oscillations; (ii) with increasing cytosolic IP3 concentration ([IP3]Cyt), the amplitude of oscillations increases (from 0.1 μM to several μM), but the frequency decreases; (iii) the frequency of [Ca2+]Cyt oscillations decreases as the ER–Mt distance increases. What is more, when the ER–Mt distance is greater than 65 nm, the μd compartment has less effect on [Ca2+]Cyt oscillations. These results suggest that Mt, μd, and IP3 can all affect the amplitude and frequency of [Ca2+]Cyt oscillations, but the mechanism is different. The model provides a comprehensive mechanism for predicting cytosolic Ca2+ concentration oscillations in mast cells, and a theoretical basis for calcium oscillations observed in mast cells, so as to better understand the regulation mechanism of calcium signaling in mast cells.

Modeling the role of endoplasmic reticulum-mitochondria microdomains in calcium dynamics

Upon inositol trisphosphate (IP3) stimulation of non-excitable cells, including vascular endothelial cells, calcium (Ca2+) shuttling between the endoplasmic reticulum (ER) and mitochondria, facilitated by complexes called Mitochondria-Associated ER Membranes (MAMs), is known to play an important role in the occurrence of cytosolic Ca2+ concentration ([Ca2+]Cyt) oscillations. A mathematical compartmental closed-cell model of Ca2+ dynamics was developed that accounts for ER-mitochondria Ca2+ microdomains as the µd compartment (besides the cytosol, ER and mitochondria), Ca2+ influx to/efflux from each compartment and Ca2+ buffering. Varying the distribution of functional receptors in MAMs vs. the rest of ER/mitochondrial membranes, a parameter called the channel connectivity coefficient (to the µd), allowed for generation of [Ca2+]Cytoscillations driven by distinct mechanisms at various levels of IP3 stimulation. Oscillations could be initiated by the transient opening of IP3 receptors facing either the cytosol or the µd, and subsequent refilling of the respective compartment by Ca2+ efflux from the ER and/or the mitochondria. Only under conditions where the µd became the oscillation-driving compartment, silencing the Mitochondrial Ca2+ Uniporter led to oscillation inhibition. Thus, the model predicts that alternative mechanisms can yield [Ca2+]Cyt oscillations in non-excitable cells, and, under certain conditions, the ER-mitochondria µd can play a regulatory role.

The Arabidopsis heterotrimeric G-protein β subunit, AGB1, is required for guard cell calcium sensing and calcium-induced calcium release.

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 Cao responses: induction of stomatal closure; inhibition of stomatal opening; [Ca2+ ]cyt oscillation; 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 of agb1 mutants and agb1/gpa1 double-mutants, as well as those of the agg1agg2Gγ double-mutant, were insensitive to Cao . These behaviors contrast with ABA-regulated stomatal movements, which involve GPA1 and AGB1/AGG3 dimers, illustrating differential partitioning of G-protein subunits among stimuli with similar ultimate impacts, which may facilitate stimulus-specific encoding. AGB1 knockouts retained reactive oxygen species and NO production, but lost YC3.6-detected [Ca2+ ]cyt oscillations in response to Cao , initiating only a single [Ca2+ ]cyt spike. Experimentally imposed [Ca2+ ]cyt oscillations restored stomatal closure in agb1. Yeast two-hybrid and bimolecular complementation fluorescence experiments revealed that AGB1 interacts with phospholipase Cs (PLCs), and Cao induced InsP3 production in Col but not in agb1. In sum, G-protein signaling via AGB1/AGG1/AGG2 is essential for Cao -regulation of stomatal apertures, and stomatal movements in response to Cao apparently require Ca2+ -induced Ca2+ release that is likely dependent on Gβγ interaction with PLCs leading to InsP3 production.

The Kinase ERULUS Controls Pollen Tube Targeting and Growth in Arabidopsis thaliana.

In this paper, we describe the role of the receptor-like kinase ERULUS (ERU) in PT growth of Arabidopsis thaliana. In silico analysis and transcriptional reporter lines revealed that ERU is only expressed in pollen and root hairs (RHs), making it a tip growth-specific kinase. Deviations from Mendelian inheritance were observed in the offspring of self-pollinated heterozygous eru plants. We found that in vivo eru PT targeting was disturbed, providing a possible explanation for the observed decrease in eru fertilization competitiveness. Extracellular calcium perception and intracellular calcium dynamics lie at the basis of in vivo pollen tube (PT) tip growth and guidance. In vitro, ERU loss-of-function lines displayed no obvious PT phenotype, unless grown on low extracellular calcium ([Ca2+]ext) medium. When grown at 12 the normal [Ca2+]ext, eru PTs grew 37% slower relative to WT PTs. Visualization of cytoplasmic [Ca2+]cyt oscillations using the Yellow Cameleon 3.6 (YC3.6) calcium sensor showed that, unlike in WT PTs, eru apical [Ca2+]cyt oscillations occur at a lower frequency when grown at lower [Ca2+]ext, consistent with the observed reduced growth velocity. Our results show that the tip growth-specific kinase ERULUS is involved in regulating Ca2+-dependent PT growth, and most importantly, fertilization efficiency through successful PT targeting to the ovules.

Live Cell Imaging with R-GECO1 Sheds Light on flg22- and Chitin-Induced Transient [Ca2+]cyt Patterns in Arabidopsis

Intracellular Ca2+ transients are an integral part of the signaling cascade during pathogen-associated molecular pattern (PAMP)-triggered immunity in plants. Yet, our knowledge about the spatial distribution of PAMP-induced Ca2+ signals is limited. Investigation of cell- and tissue-specific properties of Ca2+-dependent signaling processes requires versatile Ca2+ reporters that are able to extract spatial information from cellular and subcellular structures, as well as from whole tissues over time periods from seconds to hours. Fluorescence-based reporters cover both a broad spatial and temporal range, which makes them ideally suited to study Ca2+ signaling in living cells. In this study, we compared two fluorescence-based Ca2+ sensors: the Förster resonance energy transfer (FRET)-based reporter yellow cameleon NES-YC3.6 and the intensity-based sensor R-GECO1. We demonstrate that R-GECO1 exhibits a significantly increased signal change compared with ratiometric NES-YC3.6 in response to several stimuli. Due to its superior sensitivity, R-GECO1 is able to report flg22- and chitin-induced Ca2+ signals on a cellular scale, which allowed identification of defined [Ca2+]cyt oscillations in epidermal and guard cells in response to the fungal elicitor chitin. Moreover, we discovered that flg22- and chitin-induced Ca2+ signals in the root initiate from the elongation zone.

When isolated at full receptivity, in vitro fertilized wheat (Triticum aestivum, L.) egg cells reveal [Ca2+]cyt oscillation of intracellular origin.

During in vitro fertilization of wheat (Triticum aestivum, L.) in egg cells isolated at various developmental stages, changes in cytosolic free calcium ([Ca2+]cyt) were observed. The dynamics of [Ca2+]cyt elevation varied, reflecting the difference in the developmental stage of the eggs used. [Ca2+]cyt oscillation was exclusively observed in fertile, mature egg cells fused with the sperm cell. To determine how [Ca2+]cyt oscillation in mature egg cells is generated, egg cells were incubated in thapsigargin, which proved to be a specific inhibitor of the endoplasmic reticulum (ER) Ca2+-ATPase in wheat egg cells. In unfertilized egg cells, the addition of thapsigargin caused an abrupt transient increase in [Ca2+]cyt in the absence of extracellular Ca2+, suggesting that an influx pathway for Ca2+ is activated by thapsigargin. The [Ca2+]cyt oscillation seemed to require the filling of an intracellular calcium store for the onset of which, calcium influx through the plasma membrane appeared essential. This was demonstrated by omitting extracellular calcium from (or adding GdCl3 to) the fusion medium, which prevented [Ca2+]cyt oscillation in mature egg cells fused with the sperm. Combined, these data permit the hypothesis that the first sperm-induced transient increase in [Ca2+]cyt depletes an intracellular Ca2+ store, triggering an increase in plasma membrane Ca2+ permeability, and this enhanced Ca2+ influx results in [Ca2+]cyt oscillation.

Glucosinolate Degradation Products, Isothiocyanates, Nitriles, and Thiocyanates, Induce Stomatal Closure Accompanied by Peroxidase-Mediated Reactive Oxygen Species Production inArabidopsis thaliana

Isothiocyanates, nitriles, and thiocyanates are degradation products of glucosinolates in crucifer plants. In this study, we investigated the stomatal response to allyl isothiocyanate (AITC), 3-butenenitrile (3BN), and ethyl thiocyanate (ESCN) in Arabidopsis. AITC, 3BN, and ESCN induced stomatal closure in the wild type and the atrbohD atrbohF mutant. Stomatal closure was inhibited by catalase and salicylhydroxamic acid (SHAM). The degradation products induced extracellular reactive oxygen species (ROS) production in the rosette leaves, and intracellular ROS accumulation, NO production, and cytosolic free calcium concentration ([Ca(2+)]cyt) oscillations in guard cells, which were inhibited by SHAM. These results suggest that glucosinolate degradation products induce stomatal closure accompanied by extracellular ROS production mediated by SHAM-sensitive peroxidases, intracellular ROS accumulation, and [Ca(2+)]cyt oscillation in Arabidopsis.

Cytoplasmic Ca2+ changes dynamically during the interaction of the pollen tube with synergid cells

The directional growth of the pollen tube from the stigma to the embryo sac in the ovules is regulated by pollen-pistil interactions based on intercellular communication. Although pollen tube growth is regulated by the cytoplasmic Ca(2+) concentration ([Ca(2+)](cyt)), it is not known whether [Ca(2+)](cyt) is involved in pollen tube guidance and reception. Using Arabidopsis expressing the GFP-based Ca(2+)-sensor yellow cameleon 3.60 (YC3.60) in pollen tubes and synergid cells, we monitored Ca(2+) dynamics in these cells during pollen tube guidance and reception under semi-in vivo fertilization conditions. In the pollen tube growing towards the micropyle, pollen tubes initiated turning within 150 μm of the micropylar opening; the [Ca(2+)](cyt) in these pollen tube tips was higher than in those not growing towards an ovule in assays with myb98 mutant ovules, in which pollen tube guidance is disrupted. These results suggest that attractants secreted from the ovules affect Ca(2+) dynamics in the pollen tube. [Ca(2+)](cyt) in synergid cells did not change when the pollen tube grew towards the micropyle or entered the ovule. Upon pollen tube arrival at the synergid cell, however, [Ca(2+)](cyt) oscillation began at the micropylar pole of the synergid, spreading towards the chalazal pole. Finally, [Ca(2+)](cyt) in the synergid cell reached a maximum at pollen tube rupture. These results suggest that signals from the pollen tube induce Ca(2+) oscillations in synergid cells, and that this Ca(2+) oscillation is involved in the interaction between the pollen tube and synergid cell.

Two guard cell‐preferential MAPKs, MPK9 and MPK12, regulate YEL signalling in Arabidopsis guard cells

We report that two mitogen-activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate abscisic acid (ABA)-induced stomatal closure in Arabidopsis thaliana. Yeast elicitor (YEL) induced stomatal closure accompanied by intracellular reactive oxygen species (ROS) accumulation and cytosolic free calcium concentration ([Ca(2+) ]cyt ) oscillation. In this study, we examined whether these two MAP kinases are involved in YEL-induced stomatal closure using MAPKK inhibitors, PD98059 and U0126, and MAPK mutants, mpk9, mpk12 and mpk9 mpk12. Both PD98059 and U0126 inhibited YEL-induced stomatal closure. YEL induced stomatal closure in the mpk9 and mpk12 mutants but not in the mpk9 mpk12 mutant, suggesting that a MAPK cascade involving MPK9 and MPK12 functions in guard cell YEL signalling. However, YEL induced extracellular ROS production, intracellular ROS accumulation and cytosolic alkalisation in the mpk9, mpk12 and mpk9 mpk12 mutants. YEL induced [Ca(2+) ]cyt oscillations in both wild type and mpk9 mpk12 mutant. These results suggest that MPK9 and MPK12 function redundantly downstream of extracellular ROS production, intracellular ROS accumulation, cytosolic alkalisation and [Ca(2+) ]cyt oscillation in YEL-induced stomatal closure in Arabidopsis guard cells and are shared with ABA signalling.

Methylglyoxal-induced stomatal closure accompanied by peroxidase-mediated ROS production in Arabidopsis

Methylglyoxal (MG) is an oxygenated short aldehyde and a glycolytic intermediate that accumulates in plants under environmental stresses. Being a reactive α-oxoaldehyde, MG may act as a signaling molecule in plants during stresses. We investigated whether MG induces stomatal closure, reactive oxygen species (ROS) production, and cytosolic free calcium concentration ([Ca2+]cyt) to clarify roles of MG in Arabidopsis guard cells. MG induced production of ROS and [Ca2+]cyt oscillations, leading to stomatal closure. The MG-induced stomatal closure and ROS production were completely inhibited by a peroxidase inhibitor, salicylhydroxamic acid (SHAM), but were not affected by an NAD(P)H oxidase mutation, atrbohD atrbohF. Furthermore, the MG-elicited [Ca2+]cyt oscillations were significantly suppressed by SHAM but not by the atrbohD atrbohF mutation. Neither endogenous abscisic acid nor endogenous methyl jasmonate was involved in MG-induced stomatal closure. These results suggest that intrinsic metabolite MG can induce stomatal closure in Arabidopsis accompanied by extracellular ROS production mediated by SHAM-sensitive peroxidases, intracellular ROS accumulation, and [Ca2+]cyt oscillations.

K252a-sensitive protein kinases but not okadaic acid-sensitive protein phosphatases regulate methyl jasmonate-induced cytosolic Ca 2+ oscillation in guard cells of Arabidopsis thaliana

Methyl jasmonate (MeJA) induces stomatal closure similar to abscisic acid (ABA), and MeJA signaling in guard cells shares some signal components with ABA signaling. As part of this process, MeJA as well as ABA induce the elevation and oscillation of cytosolic free-calcium concentrations ([Ca 2+] cyt) in guard cells. While abscisic acid-induced [Ca 2+] cyt oscillation has been extensively studied, MeJA-induced [Ca 2+] cyt oscillation is less well understood. In this study, we investigated the effects of K252a (a broad-range protein kinase inhibitor) and okadaic acid (OA, a protein phosphatase 1 and 2A inhibitor) on MeJA-induced [Ca 2+] cyt oscillation in guard cells of Arabidopsis thaliana ecotype Columbia expressing the Ca 2+ reporter yellow cameleon 3.6. The protein kinase inhibitor K252a abolished MeJA-induced stomatal closure and reduced MeJA-elicited [Ca 2+] cyt oscillation. The protein phosphatase inhibitor OA, on the other hand, did not inhibit these processes. These results suggest that MeJA signaling involves activation of K252a-sensitive protein kinases upstream of [Ca 2+] cyt oscillation but not activation of an OA-sensitive protein phosphatase in guard cells of A. thaliana ecotype Columbia.

Fine-Tuning of the Cytoplasmic Ca2+ Concentration Is Essential for Pollen Tube Growth

Pollen tube growth is crucial for the delivery of sperm cells to the ovule during flowering plant reproduction. Previous in vitro imaging of Lilium longiflorum and Nicotiana tabacum has shown that growing pollen tubes exhibit a tip-focused Ca(2+) concentration ([Ca(2+)]) gradient and regular oscillations of the cytosolic [Ca(2+)] ([Ca(2+)](cyt)) in the tip region. Whether this [Ca(2+)] gradient and/or [Ca(2+)](cyt) oscillations are present as the tube grows through the stigma (in vivo condition), however, is still not clear. We monitored [Ca(2+)](cyt) dynamics in pollen tubes under various conditions using Arabidopsis (Arabidopsis thaliana) and N. tabacum expressing yellow cameleon 3.60, a fluorescent calcium indicator with a large dynamic range. The tip-focused [Ca(2+)](cyt) gradient was always observed in growing pollen tubes. Regular oscillations of the [Ca(2+)](cyt), however, were rarely identified in Arabidopsis or N. tabacum pollen tubes grown under the in vivo condition or in those placed in germination medium just after they had grown through a style (semi-in vivo condition). On the other hand, regular oscillations were observed in vitro in both growing and nongrowing pollen tubes, although the oscillation amplitude was 5-fold greater in the nongrowing pollen tubes compared with growing pollen tubes. These results suggested that a submicromolar [Ca(2+)](cyt) in the tip region is essential for pollen tube growth, whereas a regular [Ca(2+)] oscillation is not. Next, we monitored [Ca(2+)] dynamics in the endoplasmic reticulum ([Ca(2+)](ER)) in relation to Arabidopsis pollen tube growth using yellow cameleon 4.60, which has a lower affinity for Ca(2+) compared with yellow cameleon 3.60. The [Ca(2+)](ER) in pollen tubes grown under the semi-in vivo condition was between 100 and 500 microm. In addition, cyclopiazonic acid, an inhibitor of ER-type Ca(2+)-ATPases, inhibited growth and decreased the [Ca(2+)](ER). Our observations suggest that the ER serves as one of the Ca(2+) stores in the pollen tube and cyclopiazonic acid-sensitive Ca(2+)-ATPases in the ER are required for pollen tube growth.

Paracrine purinergic signaling determines lung endothelial nitric oxide production

Although the vascular bed is a major source of nitric oxide (NO) production, factors regulating the production remain unclear. We considered the role played by paracrine signaling. Determinations by fluorescence microscopy in isolated, blood-perfused rat and mouse lungs revealed that a brief lung expansion enhanced cytosolic Ca(2+) (Ca(2+)cyt) oscillations in alveolar epithelial (AEC) and endothelial (EC) cells, and NO production in EC. Furthermore, as assessed by a novel microlavage assay, alveolar ATP production increased. Intra-alveolar microinfusion of the purinergic receptor antagonist, PPADS, and the nucleotide hydrolyzing enzyme, apyrase, each completely blocked the Ca(2+)cyt and NO responses in EC. Lung expansion induced Ca(2+)cyt oscillations in mice lacking the P2Y1, but not the P2Y2, purinergic receptors, which were located in the perivascular interstitium basolateral to AEC. Prolonged lung expansion instituted by mechanical ventilation at high tidal volume increased EC expression of nitrotyrosine, indicating development of nitrosative stress in lung microvessels. These findings reveal a novel mechanism in which mechanically induced purinergic signaling couples cross-compartmental Ca(2+)cyt oscillations to microvascular NO production.

The chloroplast as a regulator of Ca2+ signalling

The chloroplast as a regulator of Ca²⁺ signalling

Are there multiple circadian clocks in plants?

We have reported that Arabidopsis might have genetically distinct circadian oscillators in multiple cell-types. Rhythms of CHLOROPHYLL A/B BINDING PROTEIN2 (CAB2) promoter activity are 2.5 h longer in phytochromeB mutants in constant red light and in cryptocrome1 cry2 double mutant (hy4-1 fha-1) in constant blue light than the wild-type. However, we found that cytosolic free Ca2+ ([Ca2+]cyt) oscillations were undetectable in these mutants in the same light conditions1. Furthermore, mutants of CIRCADIAN CLOCK ASSOCIATED1 (CCA1) have short period rhythms of leaf movement but have arrhythmic [Ca2+]cyt oscillations. More important, the timing of cab1-1 (toc1-1) mutant has short period rhythms of CAB2 promoter activity (~21 h) but, surprisingly, has a wild-type period for circadian [Ca2+]cyt oscillations (~24 h). In contrast, toc1-2, a TOC1 loss-of-function mutant, has a short period of both CAB2 and [Ca2+]cyt rhythms (~21h). Here we discuss the difference between the phenotypes of toc1-1 and toc1-2 and how rhythms of CAB2 promoter activity and circadian [Ca2+]cyt oscillations might be regulated differently.

Water Channels Are Involved in Stomatal Oscillations Encoded by Parameter‐Specific Cytosolic Calcium Oscillations

AbstractEarlier studies have shown that various stimuli can induce specific cytosolic calcium ([Ca2+]cyt) oscillations in guard cells and various oscillations in stomatal apertures. Exactly how [Ca2+]cyt oscillation signaling functions in stomatal oscillation is not known. In the present study, the epidermis of broad bean (Vicia faba L.) was used and a rapid ion‐exchange treatment with two shifting buffers differing in K+ and Ca2+ concentrations was applied. The treatment for five transients at a 10‐min transient period induced clear and regular stomatal oscillation. However, for other transient numbers and periods, the treatments induced some irregular oscillations or even no obvious oscillations in stomatal aperture. The results indicate that stomatal oscillation is encoded by parameter‐specific [Ca2+]cyt oscillation: the parameters of [Ca2+]cyt oscillation affected the occurrence rate and the parameters of stomatal oscillation. The water channel inhibitor HgCl2 completely inhibited stomatal oscillation and the inhibitory effect could be partially reversed by β‐mercaptoethanol (an agent capable of reversing water channel inhibition by HgCl2). Other inhibitory treatments against ion transport (i.e. the application of LaCl3, EGTA, or tetraethylammonium chloride (TEACI)) weakly impaired stomatal oscillation when the compounds were added after rapid ion‐exchange treatment. If these compounds were added before rapid‐ion exchange treatment, the inhibitory effect was much more apparent (except in the case of TEACI). The results of the present study suggest that water channels are involved in stomatal oscillation as a downstream element of [Ca2+]cyt oscillation signaling.(Managing editor: Wei Wang)

ATP induces alveolar/capillary cross talk in the lung

ATP released from lung epithelial cells e.g. following mechanical ventilation mediates alveolar inflammatory responses. To determine mechanisms we considered an ATP-induced signaling from alveoli to capillaries. We blood-perfused (14 ml/min) isolated rat lungs at constant arterial, venous and alveolar pressures (Palv) of 10, 3 and 5 cmH2O, respectively (n=4). We quantified cytosolic Ca2+ (Ca2+cyt) in single epithelial and endothelial cells by real-time imaging of fura 2 using epifluorescence microscopy. Under baseline conditions, Ca2+cyt oscillated in epithelial cells with an amplitude of 14±4 nM and in endothelial cells of 9±1 nM. An intra-alveolar infusion of 100 μM ATP increased the oscillation amplitude to 36±6 nM in epithelial cells and to 18±2 nM in epithelial cells (P<0.05). Both, the intra-alveolar microinfusion of the purinergic receptor antagonist, PPADS (100 μM), and the nucleotide hydrolyzing enzyme, apyrase (50 U/ml), completely blocked the alveolar ATP-induced Ca2+cyt. We interpret that alveolar ATP-induced epithelial Ca2+cyt oscillations causes a paracrine signaling to adjacent capillaries followed by endothelial Ca2+cyt oscillations. This ATP-induced alveolar/capillary cross talk may be relevant in high-tidal volume mechanical ventilation associated lung injury (supported by HL57556, HL69514).