A synthetic retinoic acid receptor γ antagonist (7C)-loaded nanoparticle enhances bone morphogenetic protein-induced bone regeneration in a rat spinal fusion model.

BACKGROUND CONTEXT: Bone morphogenetic proteins (BMPs) have potent osteoinductivity and have been applied clinically for challenging musculoskeletal conditions. However, the supraphysiological doses of BMPs used in clinical settings cause various side effects that prevent widespread use, and therefore the BMP dosage needs to be reduced. PURPOSE: To address this problem, we synthesized 7C, a retinoic acid receptor γ antagonist-loaded nanoparticle (NP), and investigated its potential application in BMP-based bone regeneration therapy using a rat spinal fusion model. STUDY DESIGN: An experimental animal study. METHODS: Fifty-three male 8-week-old Sprague-Dawley rats underwent posterolateral spinal fusion and were divided into the following five treatment groups: (1) no recombinant human (rh)BMP-2 and blank-NP (Control), (2) no rhBMP-2 and 1 µg 7C-NP (7C group), (3) low-dose rhBMP-2 (0.5 µg) and 1 µg blank-NP (L-BMP group), (4) low-dose rhBMP-2 (0.5 µg) and 1 µg 7C-NP (L-BMP + 7C group), and (5) high-dose rhBMP-2 (5.0 µg) and 1 µg blank-NP (H-BMP group). Micro-computed tomography and histologic analysis were performed 2 and 6 weeks after the surgery. RESULTS: The spinal fusion rates of the Control and 7C groups were both 0%, and those of the L-BMP, L-BMP + 7C, and H-BMP groups were 55.6%, 94.4%, and 100%, respectively. The L-BMP + 7C group markedly promoted cartilaginous tissue formation during BMP-induced endochondral bone formation that resulted in a significantly better spinal fusion rate and bone formation than in the L-BMP group. Although spinal fusion was slower in the L-BMP + 7C group, the L-BMP + 7C group formed a spinal fusion mass with better bone quality than the spinal fusion mass in the H-BMP group. CONCLUSIONS: The combined use of 7C-NP with rhBMP-2 in a rat posterolateral lumbar fusion model increased spinal fusion rate and new bone volume without deteriorating the quality of newly formed bone. CLINICAL SIGNIFICANCE: 7C-NP potentiates BMP-2-induced bone regeneration and has the potential for efficient bone regeneration with low-dose BMP-2, which can reduce the dose-dependent side effects of BMP-2 in clinical settings.

Review of Basic Research about Ossification of the Spinal Ligaments Focusing on Animal Models.

Ossification of the posterior longitudinal ligament (OPLL) is a heterotopic ossification that may cause spinal cord compression. With the recent development of computed tomography (CT) imaging, it is known that patients with OPLL often have complications related to ossification of other spinal ligaments, and OPLL is now considered part of ossification of the spinal ligaments (OSL). OSL is known to be a multifactorial disease with associated genetic and environmental factors, but its pathophysiology has not been clearly elucidated. To elucidate the pathophysiology of OSL and develop novel therapeutic strategies, clinically relevant and validated animal models are needed. In this review, we focus on animal models that have been reported to date and discuss their pathophysiology and clinical relevance. The purpose of this review is to summarize the usefulness and problems of existing animal models and to help further the development of basic research on OSL.

Entanglement of Arabidopsis Seedlings to a Mesh Substrate under Microgravity Conditions in KIBO on the ISS.

The International Space Station (ISS) provides a precious opportunity to study plant growth and development under microgravity (micro-G) conditions. In this study, four lines of Arabidopsis seeds (wild type, wild-type MCA1-GFP, mca1-knockout, and MCA1-overexpressed) were cultured on a nylon lace mesh placed on Gelrite-solidified MS-medium in the Japanese experiment module KIBO on the ISS, and the entanglement of roots with the mesh was examined under micro-G and 1-G conditions. We found that root entanglement with the mesh was enhanced, and root coiling was induced under the micro-G condition. This behavior was less pronounced in mca1-knockout seedlings, although MCA1-GFP distribution at the root tip of the seedlings was nearly the same in micro-G-grown seedlings and the ground control seedlings. Possible involvement of MCA1 in the root entanglement is discussed.

Gravity sensing in plant and animal cells.

Gravity determines shape of body tissue and affects the functions of life, both in plants and animals. The cellular response to gravity is an active process of mechanotransduction. Although plants and animals share some common mechanisms of gravity sensing in spite of their distant phylogenetic origin, each species has its own mechanism to sense and respond to gravity. In this review, we discuss current understanding regarding the mechanisms of cellular gravity sensing in plants and animals. Understanding gravisensing also contributes to life on Earth, e.g., understanding osteoporosis and muscle atrophy. Furthermore, in the current age of Mars exploration, understanding cellular responses to gravity will form the foundation of living in space.

The gravistimulation-induced very slow Ca2+ increase in Arabidopsis seedlings requires MCA1, a Ca2+-permeable mechanosensitive channel.

Gravity is a critical environmental factor affecting the morphology and function of plants on Earth. Gravistimulation triggered by changes in the gravity vector induces an increase in the cytoplasmic free calcium ion concentration ([Ca2+]c) as an early process of gravity sensing; however, its role and molecular mechanism are still unclear. When seedlings of Arabidopsis thaliana expressing apoaequorin were rotated from the upright position to the upside-down position, a biphasic [Ca2+]c-increase composed of a fast-transient [Ca2+]c-increase followed by a slow [Ca2+]c-increase was observed. We find here a novel type [Ca2+]c-increase, designated a very slow [Ca2+]c-increase that is observed when the seedlings were rotated back to the upright position from the upside-down position. The very slow [Ca2+]c-increase was strongly attenuated in knockout seedlings defective in MCA1, a mechanosensitive Ca2+-permeable channel (MSCC), and was partially restored in MCA1-complemented seedlings. The mechanosensitive ion channel blocker, gadolinium, blocked the very slow [Ca2+]c-increase. This is the first report suggesting the possible involvement of MCA1 in an early event related to gravity sensing in Arabidopsis seedlings.

A Cyclic Nucleotide-Gated Channel, HvCNGC2-3, Is Activated by the Co-Presence of Na⁺ and K⁺ and Permeable to Na⁺ and K⁺ Non-Selectively.

Cyclic nucleotide-gated channels (CNGCs) have been postulated to contribute significantly in plant development and stress resistance. However, their electrophysiological properties remain poorly understood. Here, we characterized barley CNGC2-3 (HvCNGC2-3) by the two-electrode voltage-clamp technique in the Xenopus laevis oocyte heterologous expression system. Current was not observed in X. laevis oocytes injected with HvCNGC2-3 complementary RNA (cRNA) in a bathing solution containing either Na⁺ or K⁺ solely, even in the presence of 8-bromoadenosine 3',5'-cyclic monophosphate (8Br-cAMP) or 8-bromoguanosine 3',5'-cyclic monophosphate (8Br-cGMP). A weakly voltage-dependent slow hyperpolarization-activated ion current was observed in the co-presence of Na⁺ and K⁺ in the bathing solution and in the presence of 10 µM 8Br-cAMP, but not 8Br-cGMP. Permeability ratios of HvCNGC2-3 to K⁺, Na⁺ and Cl- were determined as 1:0.63:0.03 according to reversal-potential analyses. Amino-acid replacement of the unique ion-selective motif of HvCNGC2-3, AQGL, with the canonical motif, GQGL, resulted in the abolition of the current. This study reports a unique two-ion-dependent activation characteristic of the barley CNGC, HvCNGC2-3.

Exogenous application of abscisic acid (ABA) increases root and cell hydraulic conductivity and abundance of some aquaporin isoforms in the ABA-deficient barley mutant Az34.

Background and Aims Regulation of water channel aquaporins (AQPs) provides another mechanism by which abscisic acid (ABA) may influence water flow through plants. To the best of our knowledge, no studies have addressed the changes in ABA levels, the abundance of AQPs and root cell hydraulic conductivity (LpCell) in the same tissues. Thus, we followed the mechanisms by which ABA affects root hydraulics in an ABA-deficient barley mutant Az34 and its parental line 'Steptoe'. We compared the abundance of AQPs and ABA in cells to determine spatial correlations between AQP abundance and local ABA concentrations in different root tissues. In addition, abundance of AQPs and ABA in cortex cells was related to LpCell. Methods Root hydraulic conductivity (LpRoot) was measured by means of root exudation analyses and LpCell using a cell pressure probe. The abundance of ABA and AQPs in root tissues was assessed through immunohistochemical analyses. Isoform-specific antibodies raised against HvPIP2;1, HvPIP2;2 and HvPIP2;5 were used. Key Results Immunolocalization revealed lower ABA levels in root tissues of Az34 compared with 'Steptoe'. Root hydraulic conductivity (LpRoot) was lower in Az34, yet the abundance of HvPIPs in root tissues was similar in the two genotypes. Root hair formation occurred closer to the tip, while the length of the root hair zone was shorter in Az34 than in 'Steptoe'. Application of external ABA to the root medium of Az34 and 'Steptoe' increased the immunostaining of root cells for ABA and for HvPIP2;1 and HvPIP2;2 especially in root epidermal cells and the cortical cell layer located beneath, parallel to an increase in LpRoot and LpCell. Treatment of roots with Fenton reagent, which inhibits AQP activity, prevented the ABA-induced increase in root hydraulic conductivity. Conclusion Shortly after (<2 h) ABA application to the roots of ABA-deficient barley, increased tissue ABA concentrations and AQP abundance (especially the plasma-membrane localized isoforms HvPIP2;1 and HvPIP2;2) were spatially correlated in root epidermal cells and the cortical cell layer located beneath, in conjunction with increased LpCell of the cortical cells. In contrast, long-term ABA deficiency throughout seedling development affects root hydraulics through other mechanisms, in particular the developmental timing of the formation of root hairs closer to the root tip and the length of the root hair zone.

A domain-based approach for analyzing the function of aluminum-activated malate transporters from wheat (Triticum aestivum) and Arabidopsis thaliana in Xenopus oocytes.

Wheat and Arabidopsis plants respond to aluminum (Al) ions by releasing malate from their root apices via Al-activated malate transporter. Malate anions bind with the toxic Al ions and contribute to the Al tolerance of these species. The genes encoding the transporters in wheat and Arabidopsis, TaALMT1 and AtALMT1, respectively, were expressed in Xenopus laevis oocytes and characterized electrophysiologically using the two-electrode voltage clamp system. The Al-activated currents generated by malate efflux were detected for TaALMT1 but not for AtALMT1. Chimeric proteins were generated by swapping the N- and C-terminal halves of TaALMT1 and AtALMT1 (Ta::At and At::Ta). When these chimeras were characterized in oocytes, Al-activated malate efflux was detected for the Ta::At chimera but not for At::Ta, suggesting that the N-terminal half of TaALMT1 is necessary for function in oocytes. An additional chimera, Ta(48)::At, generated by swapping 17 residues from the N-terminus of AtALMT1 with the equivalent 48 residues from TaALMT1, was sufficient to support transport activity. This 48 residue region includes a helical region with a putative transmembrane domain which is absent in AtALMT1. The deletion of this domain from Ta(48)::At led to the complete loss of transport activity. Furthermore, truncations and a deletion at the C-terminal end of TaALMT1 indicated that a putative helical structure in this region was also required for transport function. This study provides insights into the structure-function relationships of Al-activated ALMT proteins by identifying specific domains on the N- and C-termini of TaALMT1 that are critical for basal transport function and Al responsiveness in oocytes.

Calcium mobilizations in response to changes in the gravity vector in Arabidopsis seedlings: possible cellular mechanisms.

Gravity influences the growth direction of higher plants. Changes in the gravity vector (gravistimulation) immediately promote the increase in the cytoplasmic free calcium ion concentration ([Ca(2+)]c) in Arabidopsis (Arabidopsis thaliana) seedlings. When the seedlings are gravistimulated by reorientation at 180°, a transient two peaked (biphasic) [Ca(2+)]c-increase arises in their hypocotyl and petioles. Parabolic flights (PFs) can generate a variety of gravity-stimuli, and enables us to measure gravity-induced [Ca(2+)]c-increases without specimen rotation, which demonstrate that Arabidopsis seedlings possess a rapid gravity-sensing mechanism linearly transducing a wide range of gravitational changes into Ca(2+) signals on a sub-second timescale. Hypergravity by centrifugation (20 g or 300 g) also induces similar transient [Ca(2+)]c-increases. In this review, we propose models for possible cellular processes of the garavi-stimulus-induced [Ca(2+)]c-increase, and evaluate those by examining whether the model fits well with the kinetic parameters derived from the [Ca(2+)]c-increases obtained by applying gravistimulus with different amplitudes and time sequences.

Analyses of a gravistimulation-specific Ca2+ signature in Arabidopsis using parabolic flights.

Gravity is a critical environmental factor affecting the morphology and functions of organisms on the Earth. Plants sense changes in the gravity vector (gravistimulation) and regulate their growth direction accordingly. In Arabidopsis (Arabidopsis thaliana) seedlings, gravistimulation, achieved by rotating the specimens under the ambient 1g of the Earth, is known to induce a biphasic (transient and sustained) increase in cytoplasmic calcium concentration ([Ca(2+)]c). However, the [Ca(2+)]c increase genuinely caused by gravistimulation has not been identified because gravistimulation is generally accompanied by rotation of specimens on the ground (1g), adding an additional mechanical signal to the treatment. Here, we demonstrate a gravistimulation-specific Ca(2+) response in Arabidopsis seedlings by separating rotation from gravistimulation by using the microgravity (less than 10(-4)g) conditions provided by parabolic flights. Gravistimulation without rotating the specimen caused a sustained [Ca(2+)]c increase, which corresponds closely to the second sustained [Ca(2+)]c increase observed in ground experiments. The [Ca(2+)]c increases were analyzed under a variety of gravity intensities (e.g. 0.5g, 1.5g, or 2g) combined with rapid switching between hypergravity and microgravity, demonstrating that Arabidopsis seedlings possess a very rapid gravity-sensing mechanism linearly transducing a wide range of gravitational changes (0.5g-2g) into Ca(2+) signals on a subsecond time scale.

A microfluidic device to reduce treatment time of intracytoplasmic sperm injection.

OBJECTIVE: To develop a microfluidic device that can reduce the intracytoplasmic sperm injection (ICSI) treatment time by increasing sperm concentration. DESIGN: We compared the ICSI treatment time required for porcine sperm using a method employing the microfluidic device and one using the conventional microdroplet method. SETTINGS: Academic research laboratories at Okayama University. ANIMAL(S): Reproductive cells of porcine sperm, oocytes, and embryos. INTERVENTION(S): Cell manipulations, ICSI, and embryo culture. MAIN OUTCOME MEASURE(S): Average ICSI treatment time and sperm concentration. RESULT(S): The average ICSI treatment time (mean ± SEM) using the method with the microfluidic device for poor-quality semen (sperm concentration, 2.0 × 10(4) cells/mL) was significantly shorter than the treatment time using the conventional microdroplet method (265 ± 15 seconds [n = 43] vs. 347 ± 19 seconds [n = 50]). When diluted semen with a sperm concentration of 2.0 × 10(5) cells/mL was used, no significant difference was observed between the two methods (n = 50 and n = 48). CONCLUSION(S): The microfluidic device can reduce the time required for ICSI treatment that is used to increase sperm concentration in poor-quality semen samples. The results suggest that this device may be clinically useful for ICSI treatment in human assisted reproductive technology.

Expression of Arabidopsis MCA1 enhanced mechanosensitive channel activity in the Xenopus laevis oocyte plasma membrane.

Higher plants sense and respond to osmotic and mechanical stresses such as turgor, touch, flexure and gravity. Mechanosensitive (MS) channels, directly activated by tension in the cell membrane and cytoskeleton, are supposed to be involved in the cell volume regulation under hypotonic conditions and the sensing of these mechanical stresses based on electrophysiological and pharmacological studies. However, limited progress has been achieved in the molecular identification of plant MS channels. Here, we show that MCA1 (mid1-complementing activity 1; a putative mechanosensitive Ca ( 2+) -permeable channel in Arabidopsis thaliana) increased MS channel activity in the plasma membrane of Xenopus laevis oocytes. The functional and kinetic properties of MCA1 were examined by using a Xenopus laevis oocytes expression system, which showed that MCA1-dependent MS cation currents were activated by hypo-osmotic shock or by membrane stretch produced by pipette suction. Single-channel analyses suggest that MCA1 encodes a possible MS channel with a conductance of 34 pS.

Chloroplast-mediated activation of plant immune signalling in Arabidopsis.

Chloroplasts have a critical role in plant immunity as a site for the production for salicylic acid and jasmonic acid, important mediators of plant immunity. However, the molecular link between chloroplasts and the cytoplasmic-nuclear immune system remains largely unknown. Here we show that pathogen-associated molecular pattern (PAMP) signals are quickly relayed to chloroplasts and evoke specific Ca(2+) signatures in the stroma. We further demonstrate that a chloroplast-localized protein, named calcium-sensing receptor (CAS), is involved in stromal Ca(2+) transients and responsible for both PAMP-induced basal resistance and R gene-mediated hypersensitive cell death. CAS acts upstream of salicylic acid accumulation. Transcriptome analysis demonstrates that CAS is involved in PAMP-induced expression of defence genes and suppression of chloroplast gene expression possibly through (1)O(2)-mediated retrograde signalling, allowing chloroplast-mediated transcriptional reprogramming during plant immune responses. The present study reveals a previously unknown chloroplast-mediated signalling pathway linking chloroplasts to cytoplasmic-nuclear immune responses.

Inhibitory effects of methylglyoxal on light-induced stomatal opening and inward K+ channel activity in Arabidopsis.

Methylglyoxal (MG) is a reactive aldehyde derived by glycolysis. In Arabidopsis, MG inhibited light-induced stomatal opening in a dose-dependent manner. It significantly inhibited both inward-rectifying potassium (K(in)) channels in guard-cell protoplasts and an Arabidopsis K(in) channel, KAT1, heterologously expressed in Xenopus oocytes. Thus it appears that MG inhibition of stomatal opening involves MG inhibition of K(+) influx into guard cells.

Negative feedback regulation of microbe-associated molecular pattern-induced cytosolic Ca2+ transients by protein phosphorylation.

Microbe/pathogen-associated molecular patterns (MAMPs/PAMPs) often induce rises in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) and protein phosphorylation. Though they are postulated to play pivotal roles in plant innate immunity, their molecular links and the regulatory mechanisms remain largely unknown. To investigate the regulatory mechanisms for MAMP-induced Ca(2+) mobilization, we have established a transgenic rice (Oryza sativa) cell line stably expressing apoaequorin, and characterized the interrelationship among MAMP-induced changes in [Ca(2+)](cyt), production of reactive oxygen species (ROS) and protein phosphorylation. Oligosaccharide and sphingolipid MAMPs induced Ca(2+) transients mainly due to plasma membrane Ca(2+) influx, which were dramatically suppressed by a protein phosphatase inhibitor, calyculin A (CA). Hydrogen peroxide and hypo-osmotic shock triggered similar [Ca(2+)](cyt) elevations, which were not affected by CA. MAMP-induced protein phosphorylation, which is promoted by CA, has been shown to be required for ROS production and MAPK activation, while it negatively regulates MAMPs-induced Ca(2+) mobilization and may play a crucial role in temporal regulation of [Ca(2+)](cyt) signature.

An extracellular hydrophilic carboxy-terminal domain regulates the activity of TaALMT1, the aluminum-activated malate transport protein of wheat.

Al³+ -resistant cultivars of wheat (Triticum aestivum L.) release malate through the Al³+ -activated anion transport protein Triticum aestivum aluminum-activated malate transporter 1 (TaALMT1). Expression of TaALMT1 in Xenopus oocytes and tobacco suspension cells enhances the basal transport activity (inward and outward currents present in the absence of external Al³+, and generates the same Al³+ -activated currents (reflecting the Al³+-dependent transport function) as observed in wheat cells. We investigated the amino acid residues involved in this Al³+-dependent transport activity by generating a series of mutations to the TaALMT1 protein. We targeted the acidic residues on the hydrophilic C-terminal domain of TaALMT1 and changed them to uncharged residues by site-directed mutagenesis. These mutant proteins were expressed in Xenopus oocytes and their transport activity was measured before and after Al³+ addition. Three mutations (E274Q, D275N and E284Q) abolished the Al³+-activated transport activity without affecting the basal transport activity. Truncation of the hydrophilic C-terminal domain abolished both basal and Al³+-activated transport activities. Al³+-dependent transport activity was recovered by fusing the N-terminal region of TaALMT1 with the C-terminal region of AtALMT1, a homolog from Arabidopsis. These findings demonstrate that the extracellular C-terminal domain is required for both basal and Al³+-dependent TaALMT1 activity. Furthermore, we identified three acidic amino acids within this domain that are specifically required for the activation of transport function by external Al³+.

Expression of epitope-tagged proteins in plants.

Although immunoelectron microscopy is a powerful tool for visualizing the subcellular localization of target proteins, it is difficult to obtain and purify the specific antibodies required for this method. Instead of raising antibodies against individual target proteins, the use of transgenic plants expressing epitope-tagged proteins and commercially available antibodies simplifies the subcellular localization of target proteins. In this chapter, an improved method for producing transgenic plants that express epitope-tagged proteins and can be used for immunoelectron microscopic analysis is described.

Aluminium reduces sugar uptake in tobacco cell cultures: a potential cause of inhibited elongation but not of toxicity.

Aluminium is well known to inhibit plant elongation, but the role in this inhibition played by water relations remains unclear. To investigate this, tobacco (Nicotiana tabacum L.) suspension-cultured cells (line SL) was used, treating them with aluminium (50 microM) in a medium containing calcium, sucrose, and MES (pH 5.0). Over an 18 h treatment period, aluminium inhibited the increase in fresh weight almost completely and decreased cellular osmolality and internal soluble sugar content substantially; however, aluminium did not affect the concentrations of major inorganic ions. In aluminium-treated cultures, fresh weight, soluble sugar content, and osmolality decreased over the first 6 h and remained constant thereafter, contrasting with their continued increases in the untreated cultures. The rate of sucrose uptake, measured by radio-tracer, was reduced by approximately 60% within 3 h of treatment. Aluminium also inhibited glucose uptake. In an aluminium-tolerant cell line (ALT301) isogenic to SL, all of the above-mentioned changes in water relations occurred and tolerance emerged only after 6 h and appeared to involve the suppression of reactive oxygen species. Further separating the effects of aluminium on elongation and cell survival, sucrose starvation for 18 h inhibited elongation and caused similar changes in cellular osmolality but stimulated the production of neither reactive oxygen species nor callose and did not cause cell death. We propose that the inhibition of sucrose uptake is a mechanism whereby aluminium inhibits elongation, but does not account for the induction of cell death.

Closing plant stomata requires a homolog of an aluminum-activated malate transporter.

Plant stomata limit both carbon dioxide uptake and water loss; hence, stomatal aperture is carefully set as the environment fluctuates. Aperture area is known to be regulated in part by ion transport, but few of the transporters have been characterized. Here we report that AtALMT12 (At4g17970), a homolog of the aluminum-activated malate transporter (ALMT) of wheat, is expressed in guard cells of Arabidopsis thaliana. Loss-of-function mutations in AtALMT12 impair stomatal closure induced by ABA, calcium and darkness, but do not abolish either the rapidly activated or the slowly activated anion currents previously identified as being important for stomatal closure. Expressed in Xenopus oocytes, AtALMT12 facilitates chloride and nitrate currents, but not those of organic solutes. Therefore, we conclude that AtALMT12 is a novel class of anion transporter involved in stomatal closure.

Plant volatiles regulate the activities of Ca2+ -permeable channels and promote cytoplasmic calcium transients in Arabidopsis leaf cells.

A variety of plant species emit volatile compounds in response to mechanical stresses such as herbivore attack. Although these volatile compounds promote gene expression leading to anti-herbivore responses, the underlying transduction mechanisms are largely unknown. While indirect evidence suggests that the cytoplasmic free Ca(2+) concentration ([Ca(2+)](c)) plays a crucial role in the volatile-sensing mechanisms in plants, these roles have not been directly demonstrated. In the present study, we used Arabidopsis leaves expressing apoaequorin, a Ca(2+)-sensitive luminescent protein, in combination with a luminometer, to monitor [Ca(2+)](c) transients that occur in response to a variety of volatile compounds and to characterized the pharmacological properties of the increase in [Ca(2+)](c). When leaves were exposed to volatiles, [Ca(2+)](c) was transiently raised. The [Ca(2+)](c) increases induced by acyclic compounds were disrupted by Ruthenium Red, a potential plasma-membrane and endo-membrane Ca(2+)-permeable channel inhibitor, but not by 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA), an extracellular Ca(2+)-chelator, suggesting that acyclic compounds promote Ca(2+)-release from intracellular stores. On the other hand, the electrophilic compound (E)-2-hexenal promoted Ca(2+)-influx via ROS production by natural oxidation at the aquarius phase. In a gpa1-2 mutant lacking a canonical Galpha subunit, the [Ca(2+)](c) transients induced by all tested volatiles were not attenuated, suggesting that G-protein coupled receptors are not involved in the volatile-induced [Ca(2+)](c) transients in Arabidopsis leaves.