Voltage,reactive oxygen species and the influx of calcium

The apical plasma membrane of young Arabidopsis root hairs has recently been found to contain a depolarisation-activated Ca²⁺ channel, in addition to one activated by hyperpolarisation. The depolarisation-activated Ca²⁺ channel may function in signalling but the possibility that the root hair apical plasma membrane voltage may oscillate between a hyperpolarized and depolarized state suggests a role in growth control. Plant NADPH oxidase activity has yet to be considered in models of oscillatory voltage or ionic flux despite its predicted electrogenicity and voltage dependence. Activity of root NADPH oxidase was found to be stimulated by restricting Ca²⁺ influx, suggesting that these enzymes are involved in sensing Ca²⁺ entry into cells.Key words: calcium, channel, NADPH oxidase, oscillation, root hairElevation of cytosolic free Ca²⁺ ([Ca²⁺]_cyt) encodes plant cell signals.¹ Reactive oxygen species (ROS) are potent regulators of the PM Ca²⁺ channels implicated in signalling and developmental increases in [Ca²⁺]_cyt.^1,2 Plasma membrane (PM) voltage (V_m) also plays a significant part in generating specific [Ca²⁺]_cyt elevations through the opening of voltage-gated Ca²⁺-permeable channels, allowing Ca²⁺ influx.^1,3 Patch clamp electrophysiological studies on the root hair apical PM of Arabidopsis have revealed co-localisation of hyperpolarisation-activated Ca²⁺ channels (HACCs),⁴ ROS-activated HACCs⁵ and depolarisation-activated Ca²⁺ channels (DACCs).⁶ The DACC characterisation pointed to the presence of a Cl⁻-permeable conductance that was activated by moderate hyperpolarisation (−160 mV) but rapidly inactivated when the voltage was maintained at such negative values.⁶ This may be the R-type anion efflux conductance previously described in Arabidopsis root hair and root epidermal PM.⁷ Previous studies have shown that root hair PM also harbors K⁺ channels (mediating inward or outward flux)^8–10 and a H⁺-ATPase.¹¹ A key problem to address now is how these transporters interact to generate and be influenced by PM V_m, thus gating and in turn being regulated by their companion Ca²⁺ channels to encode developmental and environmental signals at the hair apex.A seminal study on the relationship between V_m and ionic fluxes in wheat root protoplasts not only confirmed oscillatory events but also determined that the PM can exist in three distinct states.¹² In the “pump state” the H⁺-ATPase predominates, there is net H⁺ efflux and the hyperpolarized V_m is negative of the equilibrium potential for K⁺ (E_K). In the “K state”, K⁺ permeability predominates but there is still net H⁺ efflux and V_m = E_K. In the third state, there is net H⁺ influx and V_m > E_K. In this depolarized H⁺-influx state, the H⁺-ATPase is thought to be inactive. Oscillations in PM V_m and H⁺ flux may be more profound in growing cells^13,14 and oscillations between these states may explain the temporal changes in H⁺ flux recently observed at the apex of growing Arabidopsis root hairs.¹⁵ Peaks of H⁺ influx may reflect a depolarized V_m that could activate DACC, suggesting that DACC would play a significant role in growth regulation. The view has arisen that the HACC would be the main driver of growth, primarily because in patch clamp assays its current is greater than DACC^4–6 and because resting V_m is usually found to be hyperpolarized. In a growing cell, with a V_m oscillating between a hyperpolarized and depolarized state, a DACC could just as well be a driver of growth given that the Ca²⁺ influx it permits could be amplified through intracellular release.The PM H⁺-ATPase traditionally lies at the core of models of voltage and ionic flux^14,16 but in terms of [Ca²⁺]_cyt regulation, the activity of PM NADPH oxidases must also now be considered. The Arabidopsis root hair apical PM also contains an NADPH oxidase (AtrbohC) that catalyses extracellular superoxide production.⁵ AtrbohC is implicated in the transition to polar growth at normal extracellular pH⁵ and also osmoregulation.¹⁷ NADPH oxidases catalyse the transport of electrons out of the cell and thus, in common with PM redox e⁻ efflux systems,¹⁸ their activity would depolarize the membrane voltage unless countered by cation efflux or anion influx.¹⁹ Two H⁺ would also be released into the cytosol for every NADPH used. The voltage-dependence of plant NADPH oxidases is unknown but e⁻ efflux by animal NADPH oxidases is fairly constant over negative V_m and decreases at very depolarized V_m.²⁰ AtrbohC is implicated in generating oscillatory ROS at the root hair apex and loss of function affects magnitude and duration of apical H⁺ flux oscillations.¹⁵ The latter suggests that AtrbohC function does in some way affect V_m, a situation extending to other root cell types (such as the epidermis) expressing NADPH oxidases.²¹NADPH oxidase activity in roots is under developmental control but also responds to anoxia and nutrient deficiency^22,23 to signal stress conditions. Blockade of PM Ca²⁺ channels by lanthanides increases superoxide production in tobacco suspension cells.²⁴ This suggests that NADPH oxidases are involved in sensing the cell''s Ca²⁺ status and the prediction would be that extracellular Ca²⁺ chelation would increase their activity. To test this, superoxide anion production by excised Arabidopsis roots was measured using reduction of the tetrazolium dye XTT (Sodium, 3′-[1-[phenylamino-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene-sulphonic acid).^25,26 Lowering extracellular Ca²⁺ from 0.5 mM to 1.4 µM by addition of 10 mM EGTA caused a mean 95% increase in diphenyliodinium-sensitive superoxide production (Fig. 1; n = 9), implicating NADPH oxidases as the source of this ROS. Stimulation of NADPH oxidase activity by decreasing Ca²⁺ influx at first appears contradictory as NADPH oxidases are stimulated by increased [Ca²⁺]_cyt²⁷ (Fig. 1). However, reduction of Ca²⁺ influx should promote voltage hyperpolarisation (just as block of K⁺ influx causes hyperpolarisation in root hairs²⁸) and this could feasibly cause increased NADPH oxidase activity. Production of superoxide could then result in ROS-activated HACC activity⁵ to increase Ca²⁺ influx.Open in a separate windowFigure 1Superoxide anion production by Arabidopsis roots. Assay medium comprised 10 mM phosphate buffer with 0.5 mM CaCl₂, 500 µM XTT, pH 6.0. Production was linear over the 30 min incubation period. Control, mean ± standard error, n = 9. Test additions were: 20 µM of the NADPH oxidase inhibitor diphenylene iodonium (DPI; n = 6); 100 µM of the Ca²⁺ ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187,³⁰ to increase [Ca²⁺]_cyt (n = 9); 10 mM of the chelator EGTA (n = 9). Dimethyl sulphoxide [DMSO; 1% (v/v)] was used as a carrier for XTT and DPI and a separate control for this is shown (n = 9).In addition to V_m, activities of PM transporters in vivo will be subject to other levels of regulation such as phosphorylation, nitrosylation and the action of [Ca²⁺]_cyt itself. Distinct spatial separation of transporters will undoubtedly play a significant role in governing V_m and [Ca²⁺]_cyt dynamics, particularly in growing cells. An NADPH oxidase has already been found sequestered in a potential PM microdomain in Medicago.²⁹ While there is still much to do on the “inventory” of PM transporters involved in Ca²⁺ signalling in any given cell, placing them in context not only requires knowledge of their genetic identity but also modelling of their concerted action.     

G蛋白调节剂和花柱S-核糖核酸酶对花粉管生长及其钙离子浓度的影响

以‘丰水’和‘幸水’梨花柱及花粉为试材,用激光共聚焦显微技术,研究了离体条件下G蛋白活性调节剂和花柱S-RNA酶对花粉管生长及其游离Ca~(2 )浓度的影响。结果表明:G蛋白激活剂CTX可促进花粉管生长,且可解除花柱S-RNA酶对自身花粉管生长的抑制作用;G蛋白抑制荆PTX和花柱S-RNA酶共同处理使异体的花粉管生长受到抑制。CTX处理使花粉管尖端区的[Ca~(2 )]_i明显升高,花柱S-RNA酶处理引起自身花粉管尖端区的[Ca~(2 )]_i梯度消失;CTX和花柱S-RNA酶共同处理则使自身花粉管内的[Ca~(2 )J_i表现出两者单独处理时的综合特征;而花柱S-RNA酶和PTX共同处理后,异体的花粉管内[Ca~(2 )]_i表现出先升高后下降的趋势。     

Regulatory volume decrease in cardiomyocytes is modulated by calcium influx and reactive oxygen species

We investigated the role of Ca²⁺ in generating reactive oxygen species (ROS) induced by hyposmotic stress (Hypo) and its relationship to regulatory volume decrease (RVD) in cardiomyocytes. Hypo-induced increases in cytoplasmic and mitochondrial Ca²⁺. Nifedipine (Nife) inhibited both Hypo-induced Ca²⁺ and ROS increases. Overexpression of catalase (CAT) induced RVD and a decrease in Hypo-induced blebs. Nife prevented CAT-dependent RVD activation. These results show a dual role of Hypo-induced Ca²⁺ influx in the control of cardiomyocyte viability. Hypo-induced an intracellular Ca²⁺ increase which activated RVD and inhibited necrotic blebbing thus favoring cell survival, while simultaneously increasing ROS generation, which in turn inhibited RVD and induced necrosis.     

Identification of hyperpolarization-activated calcium channels in apical pollen tubes of Pyrus pyrifolia

The pollen tube has been widely used to study the mechanisms underlying polarized tip growth in plants. A steep tip-to-base gradient of free cytosolic calcium ([Ca(2+)](cyt)) is essential for pollen-tube growth. Local Ca(2+) influx mediated by Ca(2+)-permeable channels plays a key role in maintaining this [Ca(2+)](cyt) gradient. Here, we developed a protocol for successful isolation of spheroplasts from pollen tubes of Pyrus pyrifolia and identified a hyperpolarization-activated cation channel using the patch-clamp technique. We showed that the cation channel conductance displayed a strong selectivity for divalent cations, with a relative permeability sequence of barium (Ba(2+)) approximately Ca(2+) > magnesium (Mg(2+)) > strontium (Sr(2+)) > manganese (Mn(2+)). This channel conductance was selective for Ca(2+) over chlorine (Cl(-)) (relative permeability P(Ca)/P(Cl) = 14 in 10 mm extracellular Ca(2+)). We also showed that the channel was inhibited by the Ca(2+) channel blockers lanthanum (La(3+)) and gadolinium (Gd(3+)). Furthermore, channel activity depended on extracellular pH and pollen viability. We propose that the Ca(2+)-permeable channel is likely to play a role in mediating Ca(2+) influx into the growing pollen tubes to maintain the [Ca(2+)](cyt) gradient.     

Reactive oxygen species produced by NADPH oxidase are involved in pollen tube growth

Tip-localized reactive oxygen species (ROS) were detected in growing pollen tubes by chloromethyl dichlorodihydrofluorescein diacetate oxidation, while tip-localized extracellular superoxide production was detected by nitroblue tetrazolium (NBT) reduction. To investigate the origin of the ROS we cloned a fragment of pollen specific tobacco NADPH oxidase (NOX) closely related to a pollen specific NOX from Arabidopsis. Transfection of tobacco pollen tubes with NOX-specific antisense oligodeoxynucleotides (ODNs) resulted in decreased amount of NtNOX mRNA, lower NOX activity and pollen tube growth inhibition. The ROS scavengers and the NOX inhibitor diphenylene iodonium chloride (DPI) inhibited growth and ROS formation in tobacco pollen tube cultures. Exogenous hydrogen peroxide (H2O2) rescued the growth inhibition caused by NOX antisense ODNs. Exogenous CaCl2 increased NBT reduction at the pollen tube tip, suggesting that Ca2+ increases the activity of pollen NOX in vivo. The results show that tip-localized ROS produced by a NOX enzyme is needed to sustain the normal rate of pollen tube growth and that this is likely to be a general mechanism in the control of tip growth of polarized plant cells.     

Lanthanum increases the rat thymocyte cytoplasmic free calcium concentration by enhancing calcium influx

In the present study, I have examined the effect of lanthanum (La3+) on cytoplasmic free calcium concentration in isolated rat thymocytes employing the quin2 technique. As with its effect on 15Ca accumulation in rat thymocytes (Segal, J. and Ingbar, S.H. (1984) Endocrinology, 115, 160-166), La3+ produced a concentration-related increase in thymocyte cytoplasmic free calcium concentration. This effect of La3+ was very prompt in onset, evident within about 30 s from the time of addition of La3+. The lowest effective concentration of La3+ was 6 microM (+22.7% above control), and it increased progressively to reach maximal values at 25 microM (+100% above control). La3+ added to quin2-loaded thymocytes suspended in a calcium-free medium was without effect. In addition, La3+ had no significant effect on 45Ca efflux, and La3+ did not inhibit calcium-ATPase activity in the rat thymocytes. These results demonstrate that in rat thymocytes La3+ increases cytoplasmic free calcium concentration by increasing the extracellular calcium influx into the cell rather than the release of calcium from an intracellular pool.     

Dual recognition of S 1 and S 4 pistils by S 4 sm pollen in self-incompatibility of Japanese pear (Pyrus pyrifolia Nakai)

Most cultivars of Japanese pear (Pyrus pyrifolia Nakai) exhibit gametophytic self-incompatibility controlled by a single S-locus with multiple S-haplotypes. A self-compatible (SC) cultivar, ??Osanijisseiki?? (S ₂ S ₄ ^sm ), arising by a bud mutation of ??Nijisseiki?? (S ₂ S ₄ ), has a stylar-part mutant S ₄ ^sm -haplotype, which lacks the pistil S ₄ gene, which is the S ₄ -RNase gene. To efficiently breed SC cultivars, we selected ??Nashi Chuukanbohon Nou 1 Gou?? (??NCN1??) harboring homozygous S ₄ ^sm from a self-progeny of Osanijisseiki and crossed it with ??Okusankichi?? (S ₅ S ₇ ), ??Hakkou?? (S ₄ S ₅ ), or ??Ri-14?? (S ₁ S ₂ ). Fruit set (%) was compared after self-pollination of the trees in the three progenies. All trees derived from the three progenies were predicted to be SC, except for the S ₄ S ₄ ^sm trees in the progeny of NCN1 × Hakkou. However, S ₁ S ₄ ^sm trees in the progeny of NCN1 × Ri-14 proved to be self-incompatible (SI). The pollen from Osanijisseiki was incompatible with ??Doitsu?? (S ₁ S ₂ ), but that from Nijisseiki was compatible, suggesting a possibility that the S ₄ ^sm pollen was rejected by S ₁ -harboring pistils. This possibility was clarified by crossing the pollen from NCN1 (S ₄ ^sm S ₄ ^sm ) to Doitsu, ??Imamuraaki?? (S ₁ S ₆ ), or ??Hougetsu?? (S ₁ S ₇ ), all of which proved incompatible. On the other hand, S ₄ ^sm pollen was accepted by pistils harboring the S ₂ , S ₃ , S ₅ , S ₆ , S ₇ , S ₉ , and S _k haplotypes. The dual recognition of S ₁ and S ₄ pistils by S ₄ ^sm pollen can be attributed to a mutation of the pollen S ₄ gene(s).     

Fast pollen tube growth in Conospermum species

BACKGROUND AND AIMS: An unusual form of pollen tube growth was observed for several Conospermum species (family Proteaceae). The rate of pollen tube growth, the number of tubes to emerge and the ultrastructure of these tubes are given here. METHODS: Pollen was germinated in vitro in different sucrose concentrations and in the presence of calcium channel blockers, and tube emergence and growth were recorded on a VCR. Measurements were taken of the number of tubes to emerge and rate of tube emergence. Pollen behaviour in vivo was also observed. The ultrastructure of germinated and ungerminated pollen was observed using TEM. RESULTS: After 10 s to 3 min in germination medium, up to three pollen tubes emerged and grew at rates of up to 55 micro m s(-1); the rate then slowed to around 2 micro m s(-1), 30 s after the initial growth spurt. Tubes were observed to grow in pulses, and the pulsed growth continued in the presence of calcium channel blockers. Optimal sugar concentration for pollen germination was 300 g L(-1), in which up to 81 % of pollen grains showed fast germination. Germination and emergence of multiple tubes were observed in sucrose concentrations of 100-800 g L(-1). The vegetative and generative nuclei moved into one of the tubes. Multiple tubes from a single grain were observed on the stigma. Under light microscopy, the cytoplasm in the tube showed a clear region at the tip. The ultrastructure of C. amoenum pollen showed a bilayered exine, with the intine being very thick at the pores, and elsewhere having large intrusions into the plasma membrane. The cytoplasm was dense with vesicles packed with inner tube cell wall material. Golgi apparatus producing secretory vesicles, and mitochondria were found throughout the tube. The tube wall was bilayered; both layers being fibrous and loosely packed. CONCLUSIONS: It is proposed that, for Conospermum, initial pollen tube wall constituents are manufactured and stored prior to pollen germination, and that tube extension occurs as described in the literature for other species, but at an exceptionally fast rate.     

Control of pollen tube tip growth by a Rop GTPase-dependent pathway that leads to tip-localized calcium influx.

We have shown that Rop1At, a pollen-specific Rop GTPase that is a member of the Rho family of small GTP binding proteins, acts as a key molecular switch controlling tip growth in Arabidopsis pollen tubes. Pollen-specific expression of constitutively active rop1at mutants induced isotropic growth of pollen tubes. Overexpression of wild-type Arabidopsis Rop1At led to ectopic accumulation of Rop1At in the plasma membrane at the tip and caused depolarization of pollen tube growth, which was less severe than that induced by the constitutively active rop1at. These results indicate that both Rop1At signaling and polar localization are critical for controlling the site of tip growth. Dominant negative rop1at mutants or antisense rop1at RNA inhibited tube growth at 0.5 mM extracellular Ca(2+), but growth inhibition was reversed by higher extracellular Ca(2+). Injection of anti-Rop antibodies disrupted the tip-focused intracellular Ca(2+) gradient known to be crucial for tip growth. These studies provide strong evidence for a Rop GTPase-dependent tip growth pathway that couples the control of growth sites with the rate of tip growth through the regulation of tip-localized extracellular Ca(2+) influxes and formation of the tip-high intracellular Ca(2+) gradient in pollen tubes.     

Presence of asparagine-linked N-acetylglucosamine and chitobiose in Pyrus pyrifolia S-RNases associated with gametophytic self-incompatibility.

S-RNases encoded by the S-locus of rosaceous and solanaceous plants discriminate between the S-alleles of pollen in gametophytic self-incompatibility reactions, but it is not clear how. We report the structures of N-glycans attached to each of the N-glycosylation sites of seven S-RNases in Pyrus pyrifolia of the Rosaceae. The structures were identified by chromatographic analysis of pyridylaminated sugar chains prepared from S4-RNase and by liquid chromatography/electrospray ionization-mass spectrometric analysis of the protease digests of reduced and S-carboxymethylated S-RNases. S4-RNase carries various types of sugar chains, including plant-specific ones with beta1-->2-linked xylose and alpha1-->3-linked fucose residues. More than 70% of the total N-glycans of S4-RNase are, however, an N-acetylglucosamine or a chitobiose (GlcNAcbeta1-->4GlcNAc), which has not been found naturally. The N-acetylglucosamine and chitobiose are mainly present at the N-glycosylation sites within the putative recognition sites of the S-RNase, suggesting that these sugar chains may interact with pollen S-product(s).     

Pollen tube growth and self-incompatibility in three Ziziphus species (Rhamnaceae)

Self-incompatibility and synchronous protandrous dichogamy have previously been reported in Ziziphus species. In this work, we conducted a comparative analysis of fluorescence microscopy observations of pollen tube growth following controlled cross pollinations of emasculated flowers and self-pollinations of non-emasculated flowers in three Ziziphus species, Z. jujuba, Z. mauritiana and Z. spina-christi, with the aim to determine the type of the self-incompatibility system of each species. In addition, to test whether autonomous self-pollination, parthenocarpy or agamospermy occurs, flowers were emasculated (or not) and covered. Fruit set and seed viability were monitored. The presence of binucleate pollen grains and the cessation of pollen tube growth in the style suggest that the self-incompatibility system operating in the studied Ziziphus species is gametophytically controlled. Controlled self-pollination in Z. mauritiana resulted in fruits that dropped off before maturation, whereas in Z. spina-christi the flowers dropped off one or two days after pollination. Following controlled self-pollination, small fruits lacking viable seeds were obtained in Z. jujuba, probably due to the stimulus provided by pollination (stimulative parthenocarpy). In the cultivar Tamar of Z. jujuba, the relatively high percentage of seedless fruits obtained in emasculated bagged flowers without hand pollination suggests that this cultivar can set seedless fruits without any pollination stimulus.     

Reactive oxygen species are involved in pollen tube initiation in kiwifruit

The role of reactive oxygen species (ROS) during pollen tube growth has been well established, but its involvement in the early germination stage is poorly understood. ROS production has been reported in germinating tobacco pollen, but evidence for a clear correlation between ROS and germination success remains elusive. Here, we show that ROS are involved in germination and pollen tube formation in kiwifruit. Using labelling with dihydrofluorescein diacetate (H(2) FDA) and nitroblue tetrazolium (NBT), endogenous ROS were detected immediately following pollen rehydration and during the lag phase preceding pollen tube emergence. Furthermore, extracellular H(2) O(2) was found to accumulate, beginning a few minutes after pollen suspension in liquid medium. ROS production was essential for kiwifruit pollen performance, since in the presence of compounds acting as superoxide dismutase/catalase mimic (Mn-5,10,15,20-tetrakis(1-methyl-4-pyridyl)21H,23H-porphin, Mn-TMPP) or as NADPH oxidase inhibitor (diphenyleneiodonium chloride, DPI), ROS levels were reduced and pollen tube emergence was severely or completely inhibited. Moreover, ROS production was substantially decreased in the absence of calcium, and by chromium and bisphenol A, which inhibit germination in kiwifruit. Peroxidase activity was cytochemically revealed after rehydration and during germination. In parallel, superoxide dismutase enzymes, particularly the Cu/Zn-dependent subtype - which function as superoxide radical scavengers - were detected by immunoblotting and by an in-gel activity assay in kiwifruit pollen, suggesting that ROS levels may be tightly regulated. Timing of ROS appearance, early localisation at the germination aperture and strict requirement for germination clearly suggest an important role for ROS in pollen grain activation and pollen tube initiation.     

Tyrosine and calcium/calmodulin kinases are common signaling components in the generation of reactive oxygen species in human lymphocytes

This study examined the signaling mechanism involved in the generation of reactive oxygen species (ROS) in human lymphocytes activated by formyl-Met-Leu-Phenylalanine (fMLP; 200 nmol/L) or phorbol-myristate-acetate (PMA; 100 nmol/L). ROS were monitored spectrophotometrically using dichlorofluorescin diacetate. fMLP and PMA significantly increased ROS above the control levels (p<0.05 and 0.001, respectively). These increases were significantly inhibited by catalase, sodium azide, and dimethylsulfoxide but not by superoxide dismutase, suggesting that the ROS apparently included hydrogen peroxide, singlet oxygen and hydroxyl ion but not superoxide anion. PMA-induced responses were reduced by tyrphostin (p<0.01), ST-638 (p<0.05), KN-62 (p<0.001), bisindolylmaleimide (p<0.001), RO-31-8220 (p<0.001), and by LY-83583 (p<0.001), suggesting significant involvement of tyrosine kinase, calcium/calmodulin kinase II, protein kinase C and guanylyl cyclase. fMLP-induced responses were significantly reduced by only tyrphostin (p<0.001), ST-638 (p<0.05), and KN-62 (p<0.01). The results show that tyrosine kinase and calcium/calmodulin kinase II are common signalling components in the production of reactive oxygen species in activated lymphocytes.     

Observations of pollen tube growth in Nicotiana alata and their implications for the mechanism of self-incompatibility

 Style squashes and stylar grafts were used to examine the growth of Nicotiana alata pollen tubes in self-compatible and self-incompatible styles. Compatible tubes typically showed a uniform layer of callose deposition in the walls and in small plugs spaced at regular intervals within the tube. Incompatible tubes were characterised by the variability of callose deposition in the walls and by larger, closer and more irregularly spaced plugs. There was no difference in the growth rate of compatible and incompatible tubes during growth through the stigma, but within the style most compatible tubes grew 20–25 mm day^-1 (maximum 30 mm day^–1), whereas incompatible tubes grew 1.0–1.5 mm day^-1 (maximum 5 mm day^–1). Many incompatible tubes continued to grow until flowers senesced, and only a small proportion died as a consequence of tip bursting. Grafting compatibly pollinated styles onto incompatible styles showed that the incompatible reaction could occur in pollen tubes between 2 and 50 mm long, and that inhibition of pollen tube growth occurred in both the upper and lower parts of the transmitting tract. Grafting incompatibly pollinated styles onto compatible styles showed that the incompatible reaction was fully reversible in at least a proportion of the pollen tubes. The findings are not consistent with the cytotoxic model of inhibition of self-pollen tubes in solanaceous plants, which assumes that the incompatible response results from the degradation of a finite amount of rRNA present in the pollen tube. However, if pollen tubes do in fact synthesise rRNA, the findings become consistent with this model. Received: 23 May 1996 / Revision accepted: 22 August 1996     

Cancer-derived immunoglobulin G promotes tumor cell growth and proliferation through inducing production of reactive oxygen species

Cancer cells have been found to express immunoglobulin G (IgG), but the exact functions and underlying mechanisms of cancer-derived IgG remain elusive. In this study, we first confirmed that downregulation of IgG restrained the growth and proliferation of cancer cells in vitro and in vivo. To elucidate its mechanism, we carried out a co-immunoprecipitation assay in HeLa cells and identified 27 potential IgG-interacting proteins. Among them, receptor of activated protein kinase C 1 (RACK1), ras-related nuclear protein (RAN) and peroxiredoxin 1 (PRDX1) are closely related to cell growth and oxidative stress, which prompted us to investigate the mechanism of action of IgG in the above phenomena. Upon confirmation of the interactions between IgG and the three proteins, further experiments revealed that downregulation of cancer-derived IgG lowered levels of intracellular reactive oxygen species (ROS) by enhancing cellular total antioxidant capacity. In addition, a few ROS scavengers, including catalase (CAT), dimethylsulfoxide (DMSO), n-acetylcysteine (NAC) and superoxide dismutase (SOD), further inhibited the growth of IgG-deficient cancer cells through suppressing mitogen-activated protein kinase/extracellular-regulated kinase (MAPK/ERK) signaling pathway induced by a low level of intracellular ROS, whereas exogenous hydrogen peroxide (H₂O₂) at low concentration promoted their survival via increasing intracellular ROS levels. Similar results were obtained in an animal model and human tissues. Taken together, our results demonstrate that cancer-derived IgG can enhance the growth and proliferation of cancer cells via inducing the production of ROS at low level. These findings provide new clues for understanding tumor proliferation and designing cancer therapy.     

Epidermal growth factor rescues trophoblast apoptosis induced by reactive oxygen species

Pre-eclampsia and intrauterine growth restriction are associated with increased apoptosis of placental villous trophoblast. This may result from placental hypoperfusion, leading to the generation of reactive oxygen species (ROS). Apoptosis can be induced in villous trophoblast following exposure to oxidative stress. Epidermal growth factor (EGF) reduces trophoblast apoptosis resulting from exposure to hypoxia. We hypothesised that exposure to hydrogen peroxide, a potent generator of ROS, would induce apoptosis in term placental villous explants and that this could be reduced by treatment with EGF. Placental explants were taken from normal term pregnancies and exposed to increasing doses of hydrogen peroxide (0–1,000 μM) or to a combination of increasing doses of hydrogen peroxide and EGF (0–100 ng/ml) for either 6 or 48 h. Apoptosis was assessed by TUNEL, proliferation by Ki-67 immunostaining, necrosis by lactate dehydrogenase activity and trophoblast differentiation by human chorionic gonadotrophin (hCG) secretion in conditioned culture media. Immunoperoxidase staining was performed to identify phosphorylated-AKT (p-AKT) and phosphorylated-PI3 kinase (p-PI3k). Exposure to 1,000 μM hydrogen peroxide for 48 h induced apoptosis in placental explants. The increase in TUNEL positive nuclei predominantly localised to syncytiotrophoblast. The amount of apoptosis was reduced to control levels by treatment with 10 and 100 ng/ml EGF. Proliferation of cytotrophoblasts within villous explants was significantly reduced following exposure to 1,000 μM hydrogen peroxide, this was restored to control levels by simultaneous treatment with 10 or 100 ng/ml EGF. Neither exposure to hydrogen peroxide or EGF altered the amount of necrosis. There was increased immunostaining for pPI3K following treatment with EGF. This study shows that apoptosis may be induced in villous trophoblast following exposure to ROS, and demonstrates the anti-apoptotic effect of EGF in trophoblast, the maintenance of which is essential for normal pregnancy.     

Pollination-induced ethylene promotes the early phase of pollen tube growth in Petunia inflata

In Petunia inflata, a species with gametophytic self-incompatibility, pollination triggers two phases of ethylene production by the pistil, the first of which peaks 3 hours after pollination with compatible or incompatible pollen. To investigate the physiological significance of the first phase of ethylene production, pollinated flowers were treated with 2,5-norbornadiene (NBD), an inhibitor of ethylene action. Treatment with NBD reduced pollen tube growth in a dose-dependent manner during the first six hours after pollination; however, pollen tube growth was insensitive to NBD if the treatment was applied 6 hours or more after pollination. Simultaneous application of exogenous ethylene substantially offset the inhibitory effects of NBD in flowers pollinated for 4 hours. Another inhibitor of ethylene action, 1-methylcyclopropene (1-MCP), also produced a strong inhibition of pollen tube growth during the first six hours of pollination. The experiments with 1-MCP pretreatment indicate that pistil tissues are the primary target of the pollination-induced ethylene.     

The effects of extracellular calmodulin on initiation of Hippeastrum rutilum pollen germination and tube growth

The effects of anti-calmodulin (CaM) serum, the CaM antagonist W7-agarose, the Ca²⁺ chelator ethyleneglycol-bis-(β-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA) and exogenous pure CaM on pollen germination and tube growth of Hippeastrum rutilum Herb were studied. Pollen germination and tube growth were inhibited or completely stopped by anti-CaM serum in a dose-dependent manner, while the same amount of preimmune serum had no effect on either process. Pollen germination and tube growth were also inhibited or completely stopped by the CaM antagonist W7-agarose and the Ca²⁺ chelator EGTA. The addition of exogenous pure CaM enhanced pollen germination and tube growth, whereas the same amount of bovine serum albumin had no effect. The inhibitory effects caused by anti-CaM serum, W7-agarose and EGTA-washing could be reversed completely by the addition of exogenous pure CaM. These results indicate that extracellular CaM initiates pollen germination and tube growth, whereas exogenous CaM enhances the above processes, and may provide a novel view for understanding the control of pollen germination and tube growth. Received: 12 December 1996 / Accepted: 15 January 1997     

Role of cytosolic free calcium in the reorientation of pollen tube growth

Growing pollen tubes of Agapanthus umbellatus exhibited a tip-to-base gradient in cytosolic free calcium ([Ca²⁺]_c). Although this gradient is believed to be involved in pollen tube growth, its role in specifying reorientation is unknown. The direction of pollen tube growth could be modified by iontophoretic micro-injection, electrical fields (EFs) or photolysis of caged Ca²⁺. Iontophoretic injection resulted in a temporary cessation of growth, an increase in [Ca²⁺]_c and, upon recovery, reoriented growth. Weak EFs also elevated [Ca²⁺]_c, reduced growth rates and resulted in the reorientation of pollen tubes towards the cathode. Treatment with very low concentrations of the Ca²⁺-channel blocker lanthanum chloride, prior to exposure to an EF, inhibited both the increase in [Ca²⁺]_c and reorientation whilst only slightly affecting growth rates. The responses of growth inhibition and reorientation were mimicked when [Ca²⁺]_c was artificially elevated by photoactivating caged Ca²⁺ (Nitr-5). Our data suggest that [Ca²⁺]_c is part of a transduction mechanism which enables growing pollen tubes to successfully reorient to directional signals in the style and thus accomplish eventual fertilization of the egg.