Nitric oxide contributes to copper tolerance by influencing ROS metabolism in Arabidopsis

Key message

Nitric oxide improves copper tolerance via modulation of superoxide and hydrogen peroxide levels. This reflects the necessity of a well-coordinated interplay between NO and ROS during stress tolerance.

Abstract

Copper (Cu) excess causes toxicity and one probable consequence of this is the disturbance of cell redox state maintenance, inter alia, by reactive oxygen- (ROS) and nitrogen species (RNS). The objective of this paper was to examine the role of nitric oxide (NO) in Cu stress tolerance and its relationship with ROS in Arabidopsis. In agar-grown seedlings, concentration-dependent Cu accumulation was observed. The 5 μM Cu resulted in reduced cell viability in the NO overproducing nox1 and gsnor1-3 root tips compared to the wild-type (WT). In contrast, 25 and 50 μM Cu caused higher viability in these mutants, while in the NO-lacking nia1nia2 lower viability was detected than in the WT. The exogenous NO donor enhanced cell viability and scavenging endogenous NO decreased it in Cu-exposed WT seedlings. Besides, SNP in nia1nia2 roots led to the improvement of viability. The ascorbic acid-deficient mutants (vtc2-1, vtc2-3) possessing slightly elevated ROS levels proved to be Cu sensitive, while miox4 showing decreased ROS production was more tolerant to Cu than the WT. In nox1 and gsnor1-3, Cu did not induce superoxide formation, and H₂O₂ accumulation occurred only in the case of NO deficiency. Based on these, under mild stress NO intensifies cell injury, while in the case of severe Cu excess it contributes to better viability. ROS were found to be responsible for aggravation of Cu-induced damage. NO alleviates acute Cu stress via modulation of O ₂ ^·? and H₂O₂ levels reflecting the necessity of a well-coordinated interplay between NO and ROS during stress tolerance.     

Examination of the superoxide/hydrogen peroxide forming and quenching potential of mouse liver mitochondria

Pyruvate dehydrogenase (PDHC) and α-ketoglutarate dehydrogenase complex (KGDHC) are important sources of reactive oxygen species (ROS). In addition, it has been found that mitochondria can also serve as sinks for cellular hydrogen peroxide (H₂O₂). However, the ROS forming and quenching capacity of liver mitochondria has never been thoroughly examined. Here, we show that mouse liver mitochondria use catalase, glutathione (GSH), and peroxiredoxin (PRX) systems to quench ROS. Incubation of mitochondria with catalase inhibitor 3-amino-1,2,4-triazole (triazole) induced a significant increase in pyruvate or α-ketoglutarate driven O₂⁻/H₂O₂ formation. 1-Choro-2,4-dinitrobenzene (CDNB), which depletes glutathione (GSH), elicited a similar effect. Auranofin (AF), a thioredoxin reductase-2 (TR2) inhibitor which disables the PRX system, did not significantly change O₂⁻/H₂O₂ formation. By contrast catalase, GSH, and PRX were all required to scavenging extramitochondrial H₂O₂. In this study, the ROS forming potential of PDHC, KGDHC, Complex I, and Complex III was also profiled. Titration of mitochondria with 3-methyl-2-oxovaleric acid (KMV), a specific inhibitor for O₂⁻/H₂O₂ production by KGDHC, induced a ~ 86% and ~ 84% decrease in ROS production during α-ketoglutarate and pyruvate oxidation. Titration of myxothiazol, a Complex III inhibitor, decreased O₂⁻/H₂O₂ formation by ~ 45%. Rotenone also lowered ROS production in mitochondria metabolizing pyruvate or α-ketoglutarate indicating that Complex I does not contribute to ROS production during forward electron transfer from NADH. Taken together, our results indicate that KGDHC and Complex III are high capacity sites for O₂⁻/H₂O₂ production in mouse liver mitochondria. We also confirm that catalase plays a role in quenching either exogenous or intramitochondrial H₂O₂.     

Reduction of hydrogen peroxide stress derived from fatty acid beta-oxidation improves fatty acid utilization in Escherichia coli

Fatty acids are a promising raw material for substance production because of their highly reduced and anhydrous nature, which can provide higher fermentation yields than sugars. However, they are insoluble in water and are poorly utilized by microbes in industrial fermentation production. We used fatty acids as raw materials for l-lysine fermentation by emulsification and improved the limited fatty acid-utilization ability of Escherichia coli. We obtained a fatty acid-utilizing mutant strain by laboratory evolution and demonstrated that it expressed lower levels of an oxidative-stress marker than wild type. The intracellular hydrogen peroxide (H₂O₂) concentration of a fatty acid-utilizing wild-type E. coli strain was higher than that of a glucose-utilizing wild-type E. coli strain. The novel mutation rpsA ^D210Y identified in our fatty acid-utilizing mutant strain enabled us to promote cell growth, fatty-acid utilization, and l-lysine production from fatty acid. Introduction of this rpsA ^D210Y mutation into a wild-type strain resulted in lower H₂O₂ concentrations. The overexpression of superoxide dismutase (sodA) increased intracellular H₂O₂ concentrations and inhibited E. coli fatty-acid utilization, whereas overexpression of an oxidative-stress regulator (oxyS) decreased intracellular H₂O₂ concentrations and promoted E. coli fatty acid utilization and l-lysine production. Addition of the reactive oxygen species (ROS) scavenger thiourea promoted l-lysine production from fatty acids and decreased intracellular H₂O₂ concentrations. Among the ROS generated by fatty-acid β-oxidation, H₂O₂ critically affected E. coli growth and l-lysine production. This indicates that the regression of ROS stress promotes fatty acid utilization, which is beneficial for fatty acids used as raw materials in industrial production.     

Catechin protection of 3T3 Swiss fibroblasts in culture under oxidative stress

The effects of catechin, a well-known in vitro antioxidant, on 3T3 Swiss fibroblasts are studied under different conditions of oxidative stress leading to cell proliferation or cytotoxicity. Various levels of reactive oxygen species (ROS), generated extracellularly by the xanthine-xanthine oxidase (X-XO) system, are at the origin of the biphasic effect on DNA synthesis by 3T3 Swiss fibroblasts. The addition of 10^?2 U XO/mL, in the absence of exogenous X, catalyzes the production of low levels of $O_2 ^{\dot - } $ and H₂O₂ in the extracellular medium, which stimulate DNA synthesis and cell division. The increase in the level of ROS, by addition of increasing X concentrations, did not enhance this effect proportionally. On the contrary, high levels of ROS inhibit DNA synthesis, the cytotoxicity induced being proportional to the level of H₂O₂ generated by the enzyme system. Catechin does not significantly modify DNA synthesis induced by low levels of ROS, but protects in a dose-dependent manner against the cytoxicity of high levels of ROS.     

Adiponectin and colon cancer: evidence for inhibitory effects on viability and migration of human colorectal cell lines

Exogenous hydrogen peroxide (H₂O₂) induces oxidative stress and apoptosis in cancer cells. This study evaluated the antiapoptotic effects of pan-caspase and caspase-3, -8, or -9 inhibitors on H₂O₂-treated Calu-6 and A549 lung cancer cells in relation to reactive oxygen species (ROS) and glutathione (GSH). Treatment with 50–500 μM H₂O₂ inhibited the growth of Calu-6 and A549 cells at 24 h and induced apoptosis in these cells. All the tested caspase inhibitors significantly prevented cell death in H₂O₂-treated lung cancer cells. H₂O₂ increased intracellular ROS levels, including that of O ₂ ^·? , at 1 and 24 h. It also increased the activity of catalase but decreased the activity of SOD. In addition, H₂O₂ triggered GSH deletion in Calu-6 and A549 cells at 24 h. It reduced GSH levels in Calu-6 cells at 1 h but increased them at 24 h. Caspase inhibitors decreased O ₂ ^·? levels in H₂O₂-treated Calu-6 cells at 1 h and these inhibitors decreased ROS levels, including that of O ₂ ^·? , in H₂O₂-treated A549 cells at 24 h. Caspase inhibitors partially attenuated GSH depletion in H₂O₂-treated A549 cells and increased GSH levels in these cells at 24 h. However, the inhibitors did not affect GSH deletion and levels in Calu-6 cells at 24 h. In conclusion, H₂O₂ induced caspase-dependent apoptosis in Calu-6 and A549 cells, which was accompanied by increases in ROS and GSH depletion. The antiapoptotic effects of caspase inhibitors were somewhat related to the suppression of H₂O₂-induced oxidative stress and GSH depletion.     

Lipoteichoic acid induces surfactant protein-A biosynthesis in human alveolar type II epithelial cells through activating the MEK1/2-ERK1/2-NF-κB pathway

Background

Asthma is a chronic inflammatory disease of the airways but recent studies have shown that alveoli are also subject to pathophysiological changes. This study was undertaken to compare hydrogen peroxide (H₂O₂) concentrations in different parts of the lung using a new technique of fractioned breath condensate sampling.

Methods

In 52 children (9-17 years, 32 asthmatic patients, 20 controls) measurements of exhaled nitric oxide (FE_NO), lung function, H₂O₂ in exhaled breath condensate (EBC) and the asthma control test (ACT) were performed. Exhaled breath condensate was collected in two different fractions, representing mainly either the airways or the alveoli. H₂O₂ was analysed in the airway and alveolar fractions and compared to clinical parameters.

Results

The exhaled H₂O₂ concentration was significantly higher in the airway fraction than in the alveolar fraction comparing each single pair (p = 0.003, 0.032 and 0.040 for the whole study group, the asthmatic group and the control group, respectively). Asthma control, measured by the asthma control test (ACT), correlated significantly with the H₂O₂ concentrations in the alveolar fraction (r = 0.606, p = 0.004) but not with those in the airway fraction in the group of children above 12 years. FE_NO values and lung function parameters did not correlate to the H₂O₂ concentrations of each fraction.

Conclusion

The new technique of fractionated H₂O₂ measurement may differentiate H₂O₂ concentrations in different parts of the lung in asthmatic and control children. H₂O₂ concentrations of the alveolar fraction may be related to the asthma control test in children.     

Auxin increases the hydrogen peroxide (H2O2) concentration in tomato (Solanum lycopersicum) root tips while inhibiting root growth

Background and Aims

The hormone auxin and reactive oxygen species (ROS) regulate root elongation, but the interactions between the two pathways are not well understood. The aim of this study was to investigate how auxin interacts with ROS in regulating root elongation in tomato, Solanum lycopersicum.

Methods

Wild-type and auxin-resistant mutant, diageotropica (dgt), of tomato (S. lycopersicum ‘Ailsa Craig’) were characterized in terms of root apical meristem and elongation zone histology, expression of the cell-cycle marker gene Sl-CycB1;1, accumulation of ROS, response to auxin and hydrogen peroxide (H₂O₂), and expression of ROS-related mRNAs.

Key Results

The dgt mutant exhibited histological defects in the root apical meristem and elongation zone and displayed a constitutively increased level of hydrogen peroxide (H₂O₂) in the root tip, part of which was detected in the apoplast. Treatments of wild-type with auxin increased the H₂O₂ concentration in the root tip in a dose-dependent manner. Auxin and H₂O₂ elicited similar inhibition of cell elongation while bringing forth differential responses in terms of meristem length and number of cells in the elongation zone. Auxin treatments affected the expression of mRNAs of ROS-scavenging enzymes and less significantly mRNAs related to antioxidant level. The dgt mutation resulted in resistance to both auxin and H₂O₂ and affected profoundly the expression of mRNAs related to antioxidant level.

Conclusions

The results indicate that auxin regulates the level of H₂O₂ in the root tip, so increasing the auxin level triggers accumulation of H₂O₂ leading to inhibition of root cell elongation and root growth. The dgt mutation affects this pathway by reducing the auxin responsiveness of tissues and by disrupting the H₂O₂ homeostasis in the root tip.     

Response characteristics of seed germination and seedling growth of Acorus tatarinowii under diesel stress

Aims

Responses of typical wetland plant Acorus tatarinowii to diesel stress were investigated to provide basis of ecological monitoring system and phytoremediation for diesel-contaminated wetland.

Methods

Greenhouse experiments were established to determine the germinability of seedlings, hydrogen peroxide in leaves, and DNA damage in roots exposed to a range of potentially phytotoxic diesel.

Results

The presence of diesel did not benefit the growth of A. tatarinowii. The germination ratio and germination rate decreased with the increase of diesel concentration, both the lowest value appeared when the concentration of diesel was 10,000 mg?kg^?1. The lowest diesel concentration (2,000 mg?kg^?1) in the soil significantly reduced the length, average diameter, and projected area of root, especially on the stress of the higher diesel concentration (4,000, 8,000, and 10,000 mg?kg^?1). Furthermore, H₂O₂ concentration in leaves rose with the increasing concentration of diesel. However, no DNA oxidative damage to root was observed in our experiment.

Conclusions

Diesel exposure significantly inhabited the seed germination, root elongation, and seedlings growth of A. tatarinowii. Diesel stress caused the accumulation of H₂O₂ in the leaves of A. tatarinowii.     

Metabolism of reactive oxygen species in cotton cytoplasmic male sterility and its restoration

To elucidate reactive oxygen species (ROS) metabolism of cotton cytoplasmic male sterility and the effects of restorer gene on the metabolism of ROS, the metabolism changes in the production and scavenging of ROS and gene expression related to ROS-scavenging enzymes were investigated in the anther mitochondria of CMS line, maintainer line and hybrid F₁. During the abortion preliminary stage (sporogenous cell division stage), anthers of CMS line had a little higher superoxide (O₂ ⁻) production rate and hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) contents than those of maintainer or hybrid F_1. Simultaneously, a little higher ROS contents might serve as a signal to increase the activity of superoxide dismutase (SOD) in anthers of CMS line to reduce the ROS damage to the anther development. But at the abortion peak (pollen mother cell meiosis stage), anthers of CMS line had extraordinarily higher ROS contents and lower ROS-scavenging enzymic activities compared with the hybrid F₁, during which the ROS contents and ROS-scavenging enzymic activities in hybrid F₁ were approximate to those of maintainer line. The expression of Mn-sod and apx mRNA in anther of CMS line was obviously inhibited when ROS produced with a great deal during anther abortion, however the gene expression in hybrid F₁ kept normal with the maintainer. Excessive accumulation of O₂^·−, H₂O₂ and MDA, significant reduction of ROS-scavenging enzymic activities and lower gene expression level of ROS-scavenging enzyme were coinstantaneous with male cells death in anthers of CMS line. But when the restorer gene was transferred into CMS line, excessive production of ROS could be eliminated in the anthers of hybrid F₁. The restorer gene likely plays an important role in keeping the dynamic balance between the production and elimination of ROS.     

Natively oxidized amino acid residues in the spinach cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript><Emphasis Type="Italic">6</Emphasis></Subscript><Emphasis Type="Italic">f</Emphasis> complex

The cytochrome b ₆ f complex of oxygenic photosynthesis produces substantial levels of reactive oxygen species (ROS). It has been observed that the ROS production rate by b ₆ f is 10–20 fold higher than that observed for the analogous respiratory cytochrome bc₁ complex. The types of ROS produced (O₂^{??, 1}O₂, and, possibly, H₂O₂) and the site(s) of ROS production within the b ₆ f complex have been the subject of some debate. Proposed sources of ROS have included the heme b _p , PQ _p ^?? (possible sources for O₂^??), the Rieske iron–sulfur cluster (possible source of O₂^?? and/or ¹O₂), Chl a (possible source of ¹O₂), and heme c _n (possible source of O₂^?? and/or H₂O₂). Our working hypothesis is that amino acid residues proximal to the ROS production sites will be more susceptible to oxidative modification than distant residues. In the current study, we have identified natively oxidized amino acid residues in the subunits of the spinach cytochrome b ₆ f complex. The oxidized residues were identified by tandem mass spectrometry using the MassMatrix Program. Our results indicate that numerous residues, principally localized near p-side cofactors and Chl a, were oxidatively modified. We hypothesize that these sites are sources for ROS generation in the spinach cytochrome b ₆ f complex.     

Apple RING finger E3 ubiquitin ligase MdMIEL1 negatively regulates salt and oxidative stresses tolerance

RING-finger-containing E3 ubiquitin ligases play important roles in plant response to biotic and abiotoc stresses. In this study, through homology analysis, a Malus× domestica MYB30-Interacting E3 Ligase 1 gene, MdMIEL1, was identified and subsequently cloned from apple ‘Gala’ (Malus×domestica). MdMIEL1 contained a zinc finger domain close to N-terminus and a RING finger domain close to Cterminus. Expression of MdMIEL1 was significantly induced by NaCl and H₂O₂ treatments. Further study demonstrated that the MdMIEL1-overexpressing Arabidopsis and apple calli were less tolerance to salt stress than wild-type control. In addition, transgenic plants had higher levels of reactive oxygen species (ROS) (H₂O₂ and O₂ ^–). And transgenic Arabidopsis and apple calli exhibited more sensitive phenotype to H₂O₂ treatment, which was associated with increased levels of ROS. These findings indicate MdMIEL1 is an important regulator involved in plant response to salt and oxidative stresses tolerance.     

Enhanced biomass and oxidative stress tolerance of <Emphasis Type="Italic">Synechococcus elongatus</Emphasis> PCC 7942 overexpressing the <Emphasis Type="Italic">DHAR</Emphasis> gene from <Emphasis Type="Italic">Brassica juncea</Emphasis>

Objectives

To improve the oxidative stress tolerance, biomass yield, and ascorbate/dehydroascorbate (AsA/DHA) ratio of Synechococcus elongatus PCC 7942 in the presence of H₂O₂, by heterologous expression of the dehydroascorbate reductase (DHAR) gene from Brassica juncea (BrDHAR).

Results

Under H₂O₂ stress, overexpression of BrDHAR in the transgenic strain (BrD) of S. elongatus greatly increased the AsA/DHA ratio. As part of the AsA recycling system, the oxidative stress response induced by reactive oxygen species was enhanced, and intracellular H₂O₂ level decreased. In addition, under H₂O₂ stress conditions, the BrD strain displayed increased growth rate and biomass, as well as higher chlorophyll content and deeper pigmentation than did wild-type and control strains.

Conclusion

By maintaining the AsA pool and redox homeostasis, the heterologous expression of BrDHAR increased S. elongatus tolerance to H₂O₂ stress, improving the biomass yield under these conditions. The results suggest that the BrD strain of S. elongatus, with its ability to attenuate the deleterious effects of ROS caused by environmental stressors, could be a promising platform for the generation of biofuels and other valuable bioproducts.

     

Oxidative reactions in axenically seedling roots of olive (Olea europaea, L.)

The production of reactive oxygen species (ROS) plays important roles in the life cycle and in the stress response and defence mechanisms of plants. Various enzyme systems are involved in the formation of ROS in the apoplast, including plasmalemma NADPH oxidase and apoplastic peroxidases. The production of O ₂ ^·? and apoplastic peroxidase and exogenous NADH oxidation activities are all strongly dependent on the age of roots??the younger the root, the greater the activity. Apoplastic production of ROS is shown in the root by using specific histochemical probes, this ROS production is growing zone dependent. In the present study, using olive seedlings, differences were also observed between cultivars, especially in O ₂ ^·? production by the Verdial cultivar which was well above that of other cultivars studied. In all the cultivars, treatment of roots with methyl jasmonate (MeJA) or methyl salicylate (MeSA) increased O ₂ ^·? production. Similar results were observed for peroxidase activity, but not for the oxidation of exogenous NADH which was either unaffected (MeJA) or even partially inhibited (MeSA). A conclusion was that MeJA or MeSA induced apoplastic production of ROS does not use exogenous NADH. Treatment with diphenylene iodonium (DPI) reduced the formation of O ₂ ^·? , but affected neither peroxidase nor NADH oxidation activities. Cyanide inhibited O ₂ ^·? production and peroxidase and NADH oxidation activities. Treatment with MnCl₂ had a strong stimulatory effect on peroxidase and NADH oxidation activities, but much less on O ₂ ^·? production. Finally, azide greatly reduced all activities, but especially O ₂ ^·? production. Together, these results indicate a relationship between oxidative activities and the processes of root growth, and that those activities are also dependent on the cultivar, as well as an involvement of peroxidases and plasmalemma NADPH oxidase in apoplast ROS production which is sensitive to DPI, azide, and cyanide but relatively insensitive to MnCl₂, while exogenous NADH oxidation is linked to peroxidase activity.     

Putrescine protects hulless barley from damage due to UV-B stress via H<Subscript>2</Subscript>S- and H<Subscript>2</Subscript>O<Subscript>2</Subscript>-mediated signaling pathways

Key message

In hulless barley, H ₂ S mediated increases in H ₂ O ₂ induced by putrescine, and their interaction enhanced tolerance to UV-B by maintaining redox homeostasis and promoting the accumulation of UV-absorbing compounds.

Abstract

This study investigated the possible relationship between putrescence (Put), hydrogen sulfide (H₂S) and hydrogen peroxide (H₂O₂) as well as the underlying mechanism of their interaction in reducing UV-B induced damage. UV-B radiation increased electrolyte leakage (EL) and the levels of malondialdehyde (MDA) and UV-absorbing compounds but reduced antioxidant enzyme activities and glutathione (GSH) and ascorbic acid (AsA) contents. Exogenous application of Put, H₂S or H₂O₂ reduced some of the above-mentioned negative effects, but were enhanced by the addition of Put, H₂S and H₂O₂ inhibitors. Moreover, the protective effect of Put against UV-B radiation-induced damage to hulless barley was diminished by dl-propargylglycine (PAG, a H₂S biosynthesis inhibitor), hydroxylamine (HT, a H₂S scavenger), diphenylene iodonium (DPI, a PM-NADPH oxidase inhibitor) and dimethylthiourea (DMTU, a ROS scavenger), and the effect of Put on H₂O₂ accumulation was abolished by HT. Taken together, as the downstream component of the Put signaling pathway, H₂S mediated H₂O₂ accumulation, and H₂O₂ induced the accumulation of UV-absorbing compounds and maintained redox homeostasis under UV-B stress, thereby increasing the tolerance of hulless barley seedlings to UV-B stress.

     

Oxidative Inactivation of Mitochondrial Aconitase Results in Iron and H2O2-Mediated Neurotoxicity in Rat Primary Mesencephalic Cultures

Background

Mitochondrial oxidative stress is a contributing factor in the etiology of numerous neuronal disorders. However, the precise mechanism(s) by which mitochondrial reactive oxygen species (ROS) modify cellular targets to induce the death of neurons remains unknown. The goal of this study was to determine if oxidative inactivation of mitochondrial aconitase (m-aconitase) resulted in the release of redox-active iron (Fe²⁺) and hydrogen peroxide (H₂O₂) and whether this contributes to cell death.

Methodology/Principal Findings

Incubation of rat primary mesencephalic cultures with the redox cycling herbicide paraquat (PQ²⁺) resulted in increased production of H₂O₂ and Fe²⁺ at times preceding cell death. To confirm the role of m-aconitase as a source of Fenton reagents and death, we overexpressed m-aconitase using an adenoviral construct thereby increasing the target available for inactivation by ROS. Co-labeling studies identified astrocytes as the predominant cell type expressing transduced m-aconitase although neurons were identified as the primary cell type dying. Oxidative inactivation of m-aconitase overexpressing cultures resulted in exacerbation of H₂O₂ production, Fe²⁺ accumulation and increased neuronal death. Increased cell death in m-aconitase overexpressing cultures was attenuated by addition of catalase and/or a cell permeable iron chelator suggesting that neuronal death occurred in part via astrocyte-derived H₂O₂.

Conclusions

These results suggest a role of ROS-sensitive m-aconitase as a source of Fe²⁺ and H₂O₂ and as a contributing factor to neurotoxicity.     

Apoplastic calmodulin promotes self-incompatibility pollen tube growth by enhancing calcium influx and reactive oxygen species concentration in Pyrus pyrifolia

Key message

This study indicated that Ca ²⁺ , ROS and actin filaments were involved with CaM in regulating pollen tube growth and providing a potential way for overcoming pear self-incompatibility.

Abstract

Calmodulin (CaM) has been associated with various physiological and developmental processes in plants, including pollen tube growth. In this study, we showed that CaM regulated the pear pollen tube growth in a concentration-dependent bi-phasic response. Using a whole-cell patch-clamp configuration, we showed that apoplastic CaM induced a hyperpolarization-activated calcium ion (Ca²⁺) current, and anti-CaM largely inhibited this type of Ca²⁺ current. Moreover, upon anti-CaM treatment, the reactive oxygen species (ROS) concentration decreased and actin filaments depolymerized in the pollen tube. Interestingly, CaM could partially rescue the inhibition of self-incompatible pear pollen tube growth. This phenotype could be mediated by CaM-enhanced pollen plasma membrane Ca²⁺ current, tip-localized ROS concentration and stabilized actin filaments. These data indicated that Ca²⁺, ROS and actin filaments were involved with CaM in regulating pollen tube growth and provide a potential way for overcoming pear self-incompatibility.