Reactive oxygen species in aerobic methane formation from vegetation

The first report of aerobic methane emissions from vegetation by an unknown mechanism¹ suggested that this potential new source may make a significant contribution to global methane emissions. We recently investigated possible mechanisms and reported^2,3 experiments in which UV-irradiation caused methane emissions from pectin, a major plant cell wall polysaccharide. Our findings also suggest that UV-generated reactive oxygen species (ROS) release methane from pectin. This has implications for all other, UV-independent processes which may generate ROS in or close to the plant cell wall and suggests a need to evaluate additional systems for ROS-generated methane emissions in leaves.Key words: methane, hydroxyl radicals, reactive oxygen species, UV, methyl esters, pectinUntil recently, the global methane budget was thought to be well understood, the only natural process for methane generation being an anaerobic microbial mechanism.⁴ However, observations by Keppler et al.¹ of aerobic methane emissions from vegetation caused controversy and called for a re-assessment of the natural sources of methane. While no mechanism was originally suggested, a putative source, the methyl ester groups of pectin, was proposed based on carbon isotope analyses.¹ We tested this hypothesis directly and reported that UV light could drive methane emissions from pectin in vitro under aerobic conditions.² While UV light was necessary for generation of methane from pectin, it is not tenable that UV was directly attacking pectic methyl ester groups since these do not absorb UV of the wavelengths used (280–400 nm). Instead, we proposed that the energy from the UV light was being absorbed by compounds such as phenolics, and that a reactive intermediary would be formed in the process. Importantly, our process had to be non-enzymic since no enzymes were present in either experimental system.^1,2 Following this hypothesis, we tested the effect of reactive oxygen species (ROS) on pectin in vitro and discovered that certain ROS cause production of methane: hydroxyl radicals (^•OH) and singlet oxygen were effective, but hydrogen peroxide and superoxide were not.³ Also, the addition of ROS-specific scavengers to pectin sheets stopped or severely reduced UV-induced methane emissions from pectin, suggesting that ROS are the intermediary in the mechanism of aerobic methane formation from pectin (Fig. 1). De-esterified pectin was produced by saponification and emitted only trace amounts of methane upon UV-irradiation, clearly establishing ester groups as the source of methane^2,3 and confirming findings of other research groups.^5,6 However, we also found that acetyl ester groups may contribute to methane emissions from pectin and should therefore be considered in future experiments attempting to identify methane sources. Interestingly, we also observed, for the first time, ethylene, ethane and CO₂ emissions from pectin upon UV-irradiation,² which corroborates the ROS hypothesis since ROS attack of methyl esters is likely to form methyl radicals, which can then either form methane or dimerise to form ethylene or ethane.Open in a separate windowFigure 1Proposed pathway for ^•OH-driven methane generation from pectin upon UV irradiation. The compound illustrated here, l-tryptophan, is merely an example of a possible photosensitiser. Hydroxyl radicals (^•OH) are shown to attack a methyl galacturonate residue of the homogalacturonan component of the pectin molecule since this is likely to be the most abundant source of methane, but the methyl esters found in xylogalacturonan domains and the acetyl esters found in homogalacturonan and rhamnogalacturonan domains are also possible methane sources. Note that only ∼70% of all galacturonic acid residues of the pectin backbone are methyl-esterified. Inset photograph shows experimental set-up during UV-irradiation of pectin.ROS are produced and destroyed constantly throughout the lifetime of plants. The generation of ROS in vivo can generally be linked to two sources: (i) a response to an external stimulus which may be perceived as a threat or (ii) a signaling process in the cell which may happen during growth, hormone action or programmed cell death.⁷ Our experiments showed that ROS could lead to methane formation from methyl ester groups; however, the origin of the ROS may not be important, only their nature. Indeed, hydrogen peroxide and superoxide, widely reported to be formed during an oxidative burst following a biotic stress,⁸ did not generate methane from pectin in vitro, and are therefore unlikely to do so in vivo. Only the hydroxyl radical (^•OH) and singlet oxygen generation led to methane formation, and therefore any process which generates them could also trigger UV-independent methane production. Abiotic stresses, such as drought, heat or salinity, which have been shown to lead to the production of ^•OH in vegetation,⁹ could therefore be processes leading to aerobic methane formation, as could exposure to elevated ozone concentrations.¹⁰ Indeed, physical injury (by cutting) of plant material has recently been demonstrated to cause methane emissions.¹¹The origin of the ROS may not be important, as long as their generation is in or close to the pectin of the plant cell wall, since ^•OH cannot travel far within a cell. Indeed, it is estimated that ^•OH typically reacts with organic matter within ∼1 nm of the site of radical production.¹² Processes such as growth^13,14 and calcium signaling,¹⁵ which both involve ROS production as an intermediary in the mechanism but are not necessarily due to external stress, may therefore have the potential to generate methane aerobically. Any process involved in the complicated pathways of ROS-regulation, for which 152 genes are responsible in Arabidopsis thaliana,¹⁶ could be involved in methane emission if the ROS generation is localised close to pectin or other potential substrates.In addition, hydrogen peroxide, which is generated in the cell walls of healthy plants,¹⁷ can be converted in the cell wall into ^•OH by processes such as the Fenton reaction,^18,19 especially in the presence of apoplastic ascorbate.^20,21 A complete analysis of the potential for ^•OH and singlet oxygen to be present in the plant cell wall is therefore necessary for a proper understanding of the different mechanisms that may drive aerobic methane generation. Further experiments into the effects of abiotic stresses other than UV on aerobic methane production from different types of vegetation are necessary in order that future in-vitro studies under simulated natural conditions can be carried out correctly. This type of study, in conjunction with direct in-vivo field studies and satellite observations, are essential to allow global estimates to be made accurately in the future and help us understand the significance of ROS-driven methane emission.     

MEK1/2 and p38-like MAP kinase successively mediate H2O2 signaling in Vicia guard cell

As a second messenger, H₂O₂ generation and signal transduction is subtly controlled and involves various signal elements, among which are the members of MAP kinase family. The increasing evidences indicate that both MEK1/2 and p38-like MAP protein kinase mediate ABA-induced H₂O₂ signaling in plant cells. Here we analyze the mechanisms of similarity and difference between MEK1/2 and p38-like MAP protein kinase in mediating ABA-induced H₂O₂ generation, inhibition of inward K⁺ currents, and stomatal closure. These data suggest that activation of MEK1/2 is prior to p38-like protein kinase in Vicia guard cells.Key words: H₂O₂ signaling, ABA, p38-like MAP kinase, MEK1/2, guard cellAn increasing number of literatures elucidate that reactive oxygen species (ROS), especially H₂O₂, is essential to plant growth and development in response to stresses,^1–4 and involves activation of various signaling events, among which are the MAP kinase cascades.^1–3,5 Typically, activation of MEK1/2 mediates NADPH oxidase-dependent ROS generation in response to stresses,^4,6–8 and the facts that MEK1/2 inhibits the expression and activation of antioxidant enzymes reveal how PD98059, the specific inhibitor of MEK1/2, abolishes abscisic acid (ABA)-induced H₂O₂ generation.^6,8,9 It has been indicated that PD98059 does not to intervene on salicylic acid (SA)-stimulated H₂O₂ signaling regardless of SA mimicking ABA in regulating stomatal closure.^2,6,8,10 Generally, activation of MEK1/2 promotes ABA-induced stomatal closure by elevating H₂O₂ generation in conjunction with inactivating anti-oxidases.Moreover, activation of plant p38-like protein kinase, the putative counterpart of yeast or mammalian p38 MAP kinase, has been reported to participate in various stress responses and ROS signaling. It has been well documented that p38 MAP kinase is involved in stress-triggered ROS signaling in yeast or mammalian cells.^11–13 Similar to those of yeast and mammals, many studies showed the activation of p38-like protein kinase in response to stresses in various plants, including Arabidopsis thaliana,^14–16 Pisum sativum,¹⁷ Medicago sativa¹⁸ and tobacco.¹⁹ The specific p38 kinase inhibitor SB203580 was found to modulate physiological processes in plant tissues or cells, such as wheat root cells,²⁰ tobacco tissue²¹ and suspension-cultured Oryza sativa cells.²² Recently, we investigate how activation of p38-like MAP kinase is involved in ABA-induced H₂O₂ signaling in guard cells. Our results show that SB203580 blocks ABA-induced stomatal closure by inhibiting ABA-induced H₂O₂ generation and decreasing K⁺ influx across the plasma membrane of Vicia guard cells, contrasting greatly with its analog SB202474, which has no effect on these events.^23,24 This suggests that ABA integrate activation of p38-like MAP kinase and H₂O₂ signaling to regulate stomatal behavior. In conjunction with SB203580 mimicking PD98059 not to mediate SA-induced H₂O₂ signaling,^23,24 these results generally reveal that the activation of p38-like MAP kinase and MEK1/2 is similar in guard cells.On the other hand, activation of p38-like MAP kinase^23,24 is not always identical to that of MEK1/2^8,25 in ABA-induced H₂O₂ signaling of Vicia guard cells. For example, H₂O₂^- and ABA-induced stomatal closure was partially reversed by SB203580. The maximum inhibition of both regent-induced stomatal closure were observed at 2 h after treatment with SB203580, under which conditions the stomatal apertures were 89% and 70% of the control values, respectively. By contrast, when PD98059 was applied together with ABA or H₂O₂, the effects of both ABA- and H₂O₂-induced stomatal closure were completely abolished (Fig. 1). These data imply that the two members of MAP kinase family are efficient in H₂O₂-stimulated stomatal closure, but p38-like MAP kinase is less susceptive than MEK1/2 to ABA stimuli.Open in a separate windowFigure 1Effects of SB203580 and PD98059 on ABA- and H₂O₂-induced stomatal closure. The experimental procedure and data analysis are according to the previous publication.^8,23,24It has been reported that ABA or NaCl activate p38 MAP kinase in the chloronema cells of the moss Funaria hygrometrica in 2∼10 min.²⁶ Similar to this, SB203580 improves H₂O₂-inhibited inward K⁺ currents after 4 min and leads it to the control level (100%) during the following 8 min (Fig. 2). However, the activation of p38-like MAP kinase in response to ABA need more time, and only recovered to 75% of the control at 8 min of treatment (Fig. 2). These results suggest that control of H₂O₂ signaling is required for the various protein kinases including p38-like MAP kinase and MEK1/2 in guard cells,^1,2,8,23,24 and the ABA and H₂O₂ pathways diverge further downstream in their actions on the K⁺ channels and, thus, on stomatal control. Other differences in action between ABA and H₂O₂ are known. For example, Köhler et al. (2001) reported that H₂O₂ inhibited the K⁺ outward rectifier in guard cells shows that H₂O₂ does not mimic ABA action on guard cell ion channels as it acts on the K⁺ outward rectifier in a manner entirely contrary to that of ABA.²⁷Open in a separate windowFigure 2Effect of SB203580 on ABA- and H₂O₂-inhibited inward K⁺ currents. The experimental procedure and data analysis are according to the previous publication.²⁴ SB203580 directs ABA- and H₂O₂-inactivated inward K⁺ currents across plasma membrane of Vicia guard cells. Here the inward K⁺ currents value is stimulated by −190 mV voltage.Based on the similarity and difference between PD98059 and SB203580 in interceding ABA and H₂O₂ signaling, we speculate the possible mechanism is that the member of MAP kinase family specially regulate signal event in ABA-triggered ROS signaling network,^1–4 and the signaling model as follows (Fig. 3).Open in a separate windowFigure 3Schematic illustration of MAP kinase-mediated H₂O₂ signaling of guard cells. The arrows indicate activation. The line indicates enhancement and the bar denotes inhibition.     

Structural basis of transmembrane domain interactions in integrin signaling

Cell surface receptors of the integrin family are pivotal to cell adhesion and migration. The activation state of heterodimeric αβ integrins is correlated to the association state of the single-pass α and β transmembrane domains. The association of integrin αIIbβ3 transmembrane domains, resulting in an inactive receptor, is characterized by the asymmetric arrangement of a straight (αIIb) and tilted (β3) helix relative to the membrane in congruence to the dissociated structures. This allows for a continuous association interface centered on helix-helix glycine-packing and an unusual αIIb(GFF) structural motif that packs the conserved Phe-Phe residues against the β3 transmembrane helix, enabling αIIb(D723)β3(R995) electrostatic interactions. The transmembrane complex is further stabilized by the inactive ectodomain, thereby coupling its association state to the ectodomain conformation. In combination with recently determined structures of an inactive integrin ectodomain and an activating talin/β complex that overlap with the αβ transmembrane complex, a comprehensive picture of integrin bi-directional transmembrane signaling has emerged.Key words: cell adhesion, membrane protein, integrin, platelet, transmembrane complex, transmembrane signalingThe communication of biological signals across the plasma membrane is fundamental to cellular function. The ubiquitous family of integrin adhesion receptors exhibits the unusual ability to convey signals bi-directionally (outside-in and inside-out signaling), thereby controlling cell adhesion, migration and differentiation.^1–5 Integrins are Type I heterodimeric receptors that consist of large extracellular domains (>700 residues), single-pass transmembrane (TM) domains, and mostly short cytosolic tails (<70 residues). The activation state of heterodimeric integrins is correlated to the association state of the TM domains of their α and β subunits.^6–10 TM dissociation initiated from the outside results in the transmittal of a signal into the cell, whereas dissociation originating on the inside results in activation of the integrin to bind ligands such as extracellular matrix proteins. The elucidation of the role of the TM domains in integrin-mediated adhesion and signaling has been the subject of extensive research efforts, perhaps commencing with the demonstration that the highly conserved GFFKR sequence motif of α subunits (Fig. 1), which closely follows the first charged residue on the intracellular face, αIIb(K989), constrains the receptor to a default low affinity state.¹¹ Despite these efforts, an understanding of this sequence motif had not been reached until such time as the structure of the αIIb TM segment was determined.¹² In combination with the structure of the β3 TM segment¹³ and available mutagenesis data,^6,9,10,14,15 this has allowed the first correct prediction of the overall association of an integrin αβ TM complex.¹² The predicted association was subsequently confirmed by the αIIbβ3 complex structure determined in phospholipid bicelles,¹⁶ as well as by the report of a similar structure based on molecular modeling using disulfide-based structural constraints.¹⁷ In addition to the structures of the dissociated and associated αβ TM domains, their membrane embedding was defined^{12,13,16,18,19} and it was experimentally recognized that, in the context of the native receptor, the TM complex is stabilized by the inactive, resting ectodomain.¹⁶ These advances in integrin membrane structural biology are complemented by the recent structures of a resting integrin ectodomain and an activating talin/β cytosolic tail complex that overlap with the αβ TM complex,^20,21 allowing detailed insight into integrin bi-directional TM signaling.Open in a separate windowFigure 1Amino acid sequence of integrin αIIb and β3 transmembrane segments and flanking regions. Membrane-embedded residues^{12,13,16,18,19} are enclosed by a gray box. Residues 991–995 constitute the highly conserved GFFKR sequence motif of integrin α subunits.     

Compatible solutes mitigate damaging effects of salt stress by reducing the impact of stress-induced reactive oxygen species

Under abiotic stress conditions, rapid increases in reactive oxygen species (ROS) levels occurs within plant cells. Although their role as a major signalling agent in plants is now acknowledged, elevated ROS levels can result in an impairment of membrane integrity. Similar to our previous findings on imposition of salt stress, application of the hydroxyl radical (OH^•) to Arabidopsis roots results in a massive efflux of K⁺ from epidermal cells. This is likely to cause significant damage to cell metabolism. Since K⁺ loss also occurs after salt application and salt stress leads to increased cellular ROS levels, we suggest that at least some of the detrimental effects of salinity is due to damage by its resulting ROS on K⁺ homeostasis. We also observed a comparative reduction in K⁺ efflux by compatible solutes after both oxidative and salt stress. Thus, we propose that under saline conditions, compatible solutes mitigate the oxidative stress damage to membrane transporters. Whether this amelioration is due to free-radical scavenging or by direct protection of transporter systems, warrants further investigation.Key words: compatible solutes, hydroxyl radical, potassium efflux, reactive oxygen species, salt stressReactive oxygen species (ROS) are continuously produced as by-products of various metabolic pathways.¹ Under unstressed steady-state conditions, cellular ROS levels are kept in check by the sophisticated antioxidant defence system.² However, under adverse environmental conditions, the balance between ROS production and its subsequent scavenging may be perturbed, leading to a rapid increase in ROS levels.³ Although significant progress has been made in defining ROS as a major signalling agent in plants,³ ROS can react with a large variety of biomolecules, causing lipid peroxidation and impairing membrane integrity.^4,5 One such abiotic stress is salt stress,⁶ with ROS generation occurring within minutes of salt application.⁷ Alleviation of oxidative damage may be, therefore, an important strategy of plant salt tolerance.⁸One of the earliest measurable responses to salt stress is a massive K⁺ efflux from plant roots.^9,10 Such K⁺ efflux is initiated within seconds of acute salt stress and may last for several hours^11,12 reducing the intracellular K⁺ pool^13,14 and significantly impairing cell metabolism. Consistent with the key role of K⁺ homeostasis in salt tolerance mechanisms¹⁵ a reduction of K⁺ efflux correlates with increased salt tolerance.^11,12We have previously reported that hydroxyl radical (OH^•) application to Arabidopsis roots also results in a rapid efflux of K⁺ from the epidermis.¹⁶ In this report, we find a similar K⁺ efflux response.¹⁷ As is the case for salt stress,⁹ we found that membrane depolarisation could be responsible for a substantial part of this efflux. However, an observed discrepancy between the membrane depolarisation and the pattern of K⁺ efflux indicates that voltage-dependence is not the only factor influencing K⁺ loss from the root cells after oxidative stress. Demidchik et al.¹⁶ demonstrated that stress-induced K⁺ efflux could be mediated by activation of K⁺ outward rectifying channels directly by OH^•. This direct effect on K⁺ transporters could also account for our observed delay before the peak efflux of K⁺ is measured, indicating that a certain amount of time is required before maximal direct damage by OH^• to transporters occurs. Because both K⁺ channel blockers and non-selective cation channel blockers reduce this efflux, it indicates non-specificity in OH^• attack. Furthermore, combinations of these channel blockers were effective in reducing K⁺ efflux implying that, at least in the short term, the damaging effects of OH^• is due to compromising the transporter systems as opposed to lipid peroxidation. Certainly, K⁺ channels harbour reactive groups, thus are expected to be sensitive to ROS.¹⁸We have previously shown that the exogenous application of low concentrations of a variety of compatible solutes reduces the salt-induced K⁺ efflux.^19,20 Plants, when confronted with a saline environment, respond with a significant elevation in their compatible solute levels. This ameliorates the detrimental effects of salinity.²¹ However, their original proposed role in cellular osmoregulation is under question: their concentration in transgenic plants overexpressing osmolyte biosynthetic genes is not significant for osmotic adjustment, despite showing improved salt tolerance.⁸ Furthermore, one hallmark of the detoxification effect is its lack of specificity, that is, transgenic plants have increased tolerance not only to high salt, but also to drought, cold and heat shock,^22,23 stresses that also result in ROS production.³ Certainly, ecotopic expression studies suggest that compatible solutes increase stress tolerance by protection of membranes and proteins against ROS.⁶We show that in this work that exogenous application of low concentrations of a range of compatible solutes significantly reduces OH^•-induced K⁺ efflux,¹⁷ a similar effect to that we reported after salt application to barley roots¹⁹ and also observed in Arabidopsis (Fig. 1). Interestingly, we found that not only known free-radical scavenging osmolytes,²⁴ but also glycine betaine, previously found to be non-effective in ROS scavenging,²⁴ were effective in reducing OH^•-induced K⁺ efflux. Indeed, glycine betaine showed a greater mitigation of OH^•-induced K⁺ efflux compared to that induced by 50 mM NaCl (Fig. 1). However, it is open to speculation as to whether this mitigation is via direct channel blocking, a direct protection of ion channel proteins or by some other protective mechanism.Open in a separate windowFigure 1Effects of exogenous supply of compatible solutes on net peak K⁺ efflux after application of either 1 mM Cu/a or 50 mM NaCl. Roots were preincubated for 1 h in 5 mM concentration of a number of compatible solutes prior to treatment, Mean ± SE (n = 6-8).In our further investigations we have found that salt-tolerant barley show a reduced ROS-induced K⁺ efflux compared to sensitive varieties.²⁵ This superior ability of salt-tolerant barley cultivars of preventing K⁺ loss further indicates a possible causal link between salt and oxidative stress tolerance. We propose that upon the imposition of salt stress, the instantaneously resulting membrane depolarisation9 results in activation of depolarisation activated K⁺ outward-rectifying channels, leading to the initial massive K⁺ efflux. Over the longer term, ROS levels within the plant cell increase,⁷ resulting in direct damage to K⁺ transporters and the longer-term sustained loss of K⁺ from the cell. Due to mitigation of both NaCl- and OH^•-induced K⁺ efflux by compatible solutes, we propose that one of their primary amelioratory effects is through reducing the damaging effects of salt-produced ROS on K⁺ transporter, and by this means, reducing the effects of stress damage. Whether this amelioration is achieved through free-radical scavenging or due to a direct protection of membrane transports warrants further investigation.     

Plant defensins: Defense,development and application

Plant defensins are small, highly stable, cysteine-rich peptides that constitute a part of the innate immune system primarily directed against fungal pathogens. Biological activities reported for plant defensins include antifungal activity, antibacterial activity, proteinase inhibitory activity and insect amylase inhibitory activity. Plant defensins have been shown to inhibit infectious diseases of humans and to induce apoptosis in a human pathogen. Transgenic plants overexpressing defensins are strongly resistant to fungal pathogens. Based on recent studies, some plant defensins are not merely toxic to microbes but also have roles in regulating plant growth and development.Key words: defensin, antifungal, antimicrobial peptide, development, innate immunityDefensins are diverse members of a large family of cationic host defence peptides (HDP), widely distributed throughout the plant and animal kingdoms.^1–3 Defensins and defensin-like peptides are functionally diverse, disrupting microbial membranes and acting as ligands for cellular recognition and signaling.⁴ In the early 1990s, the first members of the family of plant defensins were isolated from wheat and barley grains.^5,6 Those proteins were originally called γ-thionins because their size (∼5 kDa, 45 to 54 amino acids) and cysteine content (typically 4, 6 or 8 cysteine residues) were found to be similar to the thionins.⁷ Subsequent “γ-thionins” homologous proteins were indentified and cDNAs were cloned from various monocot or dicot seeds.⁸ Terras and his colleagues⁹ isolated two antifungal peptides, Rs-AFP1 and Rs-AFP2, noticed that the plant peptides'' structural and functional properties resemble those of insect and mammalian defensins, and therefore termed the family of peptides “plant defensins” in 1995. Sequences of more than 80 different plant defensin genes from different plant species were analyzed.¹⁰ A query of the UniProt database (www.uniprot.org/) currently reveals publications of 371 plant defensins available for review. The Arabidopsis genome alone contains more than 300 defensin-like (DEFL) peptides, 78% of which have a cysteine-stabilized α-helix β-sheet (CSαβ) motif common to plant and invertebrate defensins.¹¹ In addition, over 1,000 DEFL genes have been identified from plant EST projects.¹²Unlike the insect and mammalian defensins, which are mainly active against bacteria,^2,3,10,13 plant defensins, with a few exceptions, do not have antibacterial activity.¹⁴ Most plant defensins are involved in defense against a broad range of fungi.^2,3,10,15 They are not only active against phytopathogenic fungi (such as Fusarium culmorum and Botrytis cinerea), but also against baker''s yeast and human pathogenic fungi (such as Candida albicans).² Plant defensins have also been shown to inhibit the growth of roots and root hairs in Arabidopsis thaliana¹⁶ and alter growth of various tomato organs which can assume multiple functions related to defense and development.⁴     

Reactive oxygen species as transducers of sphinganine-mediated cell death pathway

Long chain bases or sphingoid bases are building blocks of complex sphingolipids that display a signaling role in programmed cell death in plants. So far, the type of programmed cell death in which these signaling lipids have been demonstrated to participate is the cell death that occurs in plant immunity, known as the hypersensitive response. The few links that have been described in this pathway are: MPK6 activation, increased calcium concentrations and reactive oxygen species (ROS) generation. The latter constitute one of the more elusive loops because of the chemical nature of ROS, the multiple possible cell sites where they can be formed and the ways in which they influence cell structure and function.Key words: hydrogen peroxide, long chain bases, programmed cell death, reactive oxygen species, sphinganine, sphingoid bases, superoxideA new transduction pathway that leads to programmed cell death (PCD) in plants has started to be unveiled.^1,2 Sphingoid bases or long chain bases (LCBs) are the distinctive elements in this PCD route that naturally operates in the entrance site of a pathogen as a way to contend its spread in the plant tissues.^2,3 This defense strategy has been known as the hypersensitive response (HR).^4,5As a lately discovered PCD signaling circuit, three connected transducers have been clearly identified in Arabidopsis: the LCB sphinganine (also named dihydrosphingosine or d18:0); MPK6, a mitogen activated kinase and superoxide and hydrogen peroxide as reactive oxygen species (ROS).^1,2 In addition, calcium transients have been recently allocated downstream of exogenously added sphinganine in tobacco cells.⁶Contrary to the signaling lipids derived from complex glycerolipid degradation, sphinganine, a metabolic precursor of complex sphingolipids, is raised by de novo synthesis in the endoplasmic reticulum to mediate PCD.^1,2 Our recent work demonstrated that only MPK6 and not MPK3 (commonly functionally redundant kinases) acts in this pathway and is positioned downstream of sphinganine elevation.² Although ROS have been identified downstream of LCBs in the route towards PCD,¹ the molecular system responsible for this ROS generation, their cellular site of formation and their precise role in the pathway have not been unequivocally identified. ROS are produced in practically all cell compartments as a result of energy transfer reactions, leaks from the electron transport chains, and oxidase and peroxidase catalysis.⁷Similar to what is observed in pathogen defense,³ increases in endogenous LCBs may be elicited by addition of fumonisin B1 (FB1) as well; FB1 is a mycotoxin that inhibits ceramide synthase. This inhibition results in an accumulation of its substrate, sphinganine and its modified forms, leading to the activation of PCD.^1,2,8 The application of FB1 is a commonly used approach for the study of PCD elicitation in Arabidopsis.^1,2,9–11An early production of ROS has been linked to an increase of LCBs. For example, an H₂O₂ burst is found in tobacco cells after 2–20 min of sphinganine supplementation,¹² and superoxide radical augmented in the medium 60 min after FB1 or sphinganine addition to Arabidopsis protoplasts (Fig. 1A). In consonance with this timing, both superoxide and H₂O₂ were detected in Arabidopsis leaves after 3–6 h exposure to FB1 or LCBs.¹ However, the source of ROS generation associated with sphinganine elevation seems to not be the same in both species: in tobacco cells, ROS formation is apparently dependent on a NADPH oxidase activity, a ROS source consistently implicated in the HR,^13,14 while in Arabidopsis, superoxide formation was unaffected by diphenyliodonium (DPI), a NADPH oxidase inhibitor (Fig. 1A). It is possible that the latter oxidative burst is due to an apoplastic peroxidase,¹⁵ or to intracellular ROS that diffuse outwards.^16,17 These results also suggest that both tobacco and Arabidopsis cells could produce ROS from different sources.Open in a separate windowFigure 1ROS are produced at early and long times in the FB1-induced PCD in Arabidopsis thaliana (Col-0). (A) Superoxide formation by Arabidopsis protoplasts is NADPH oxidase-independent and occurs 60 min after FB1 or sphinganine (d18:0) exposure. Protoplasts were obtained from a cell culture treated with cell wall lytic enzymes. Protoplasts were incubated with 10 µM FB1 or 10 µM sphinganine for 1 h. Then, cells were vacuum-filtered and the filtrate was used to determine XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, disodium salt] reduction as described in references ²⁸ and ²⁹. DPI was used at 50 µM. (B) H₂O₂ formation in Arabidopsis wt and lcb2a-1 mutant in the presence and absence of FB1. Arabidopsis seedlings were exposed to 10 µM FB1 and after 48 h seedlings were treated with DA B (3,3-diaminobencidine) to detect H₂O₂ according to Thordal-Christensen et al.³⁰It has been suggested that the H₂O₂ burst associated with the sphinganine signaling pathway leads to the expression of defense-related genes but not to the PCD itself in tobacco cells.¹² It is possible that ROS are involved in the same way in Arabidopsis, since defense gene expression is also induced by FB1 in Arabidopsis.⁹ In this case, it will be important to define how the early ROS that are DPI-insensitive could contribute to the PCD manifestation mediated by sphinganine.The generation of ROS (4–60 min) found in Arabidopsis was associated to three conditions: the addition of sphinganine (Fig. 1A), FB1 (Fig. 1A) or pathogen elicitors.¹⁵ This is consistent with the MPK6 activation time, which is downstream of sphinganine elevation and occurs as early as 15 min of FB1 or sphinganine exposure.² All of them are events that appear as initial steps in the relay pathway that produces PCD.In order to explore a possible participation of ROS at more advanced times of PCD progression, we detected in situ H₂O₂ formation in Arabidopsis seedlings previously exposed to FB1 for 48 h. As shown in Figure 1B, formation of the brown-reddish precipitate corresponding to the reaction of H₂O₂ with 3,3′-diaminobenzidine (DAB) was only visible in the FB1-exposed wild type plants, as compared to the non-treated plants. However, when lcb2a-1 mutant seedlings were used, FB1 exposure had a subtle effect in ROS formation. This mutant has a T-DNA insertion in the gene encoding subunit LCB2a from serine palmitoyltransferase (SPT), which catalyzes the first step in sphingolipid synthesis¹⁸ and the mutant has a FB1-resistant phenotype.² These results indicate that mutations in the LCB1¹ and LCB2a² genes (coding for the subunits of the heterodimeric SPT) that lead to a non-PCD phenotype upon the FB1 treatment, are unable to produce H₂O₂. In addition, they suggest that high levels of hydrogen peroxide are produced at advanced times in the PCD mediated by LCBs in Arabidopsis.Exposure of Arabidopsis to an avirulent strain of Pseudomonas syringae produces an endogenous elevation of LCBs as a way to implement defense responses that include HR-PCD.³ In this condition, we clearly detected H₂O₂ formation inside chloroplasts (Fig. 2A). When ultrastructure of the seedlings tissues exposed to FB1 for 72 h was analyzed, integrity of the chloroplast membrane system was severely affected in Arabidopsis wild-type seedlings exposed to FB1.² Therefore, we suggest that ROS generation-LCB induced in the chloroplast could be responsible of the observed membrane alteration, as noted by Liu et al. who found impairment in chloroplast function as a result of H₂O₂ formation in this organelle from tobacco plants. Interestingly, these plants overexpressed a MAP kinase kinase that activated the kinase SIPK, which is the ortholog of the MPK6 from Arabidopsis, a transducer in the PCD instrumented by LCBs.²Open in a separate windowFigure 2Conditions of LCBs elevation produce H₂O₂ formation in the chloroplast and perturbation in the membrane morphology of mitochondria. (A) Exposure of Arabidopsis leaves to the avirulent strain Pseudomonas syringae pv. tomato DC3000 (avrRPM1) (or Pst avrRPM1) induces H₂O₂ formation in the chloroplast. Arabidopsis leaves were infiltrated with 1 × 10⁸ UFC/ml Pst avrRPM1 and after 18 h, samples were treated to visualize H₂O₂ formation with the DAB reaction. Controls were infiltrated with 10 mM MgCl₂ and then processed for DAB staining. Then, samples were analyzed in an optical photomicroscope Olympus Provis Model AX70. (B) Effect of FB1 on mitochondria ultrastructure. Wild type Arabidopsis seedlings were treated with FB1 for 72 h and tissues were processed and analyzed according to Saucedo et al.² Ch, chloroplast; M, mitochondria; PM, plasma membrane. Arrows show mitochondrial cisternae. Bars show the correspondent magnification.In addition, we have detected alterations in mitochondria ultrastructure as a result of 72 h of FB1 exposure (Fig. 2B). These alterations mainly consist in the reduced number of cristae, the membrane site of residence of the electron transport complexes. In this sense, it has been shown that factors that induce PCD such as the victorin toxin, methyl jasmonate and H₂O₂ produce alterations in mitochondrial morphology.^20–22 In fact, some of these studies propose that ROS are formed in the mitochondria and then diffuse to the chloroplasts.^22–24It is reasonable to envisage that damage of the membrane integrity of these two organelles reflects the effects of vast amounts of ROS produced by the electron transport chains.^25,26 Recent evidence supports the destruction of the photosynthetic apparatus associated to the generation of ROS in the HR.²⁶ At this time of PCD progression, ROS could be contributing to shut down the energy machinery in the cell, which ultimately would become the point of no-return of PCD²⁷ as part of the execution program of the cell death mediated by LCBs.In conclusion, we propose that ROS can display two different functional roles in the PCD process driven by LCBs. These roles depend on the time of ROS expression, the cellular site where they are generated, the enzymes that produce them, and the magnitude in which they are formed.     

Nitric oxide production is not required for dihydrosphingosine-induced cell death in tobacco BY-2 cells

Sphinganine or dihydrosphingosine (d18:0, DHS), one of the most abundant free sphingoid Long Chain Base (LCB) in plants, is known to induce a calcium-dependent programmed cell death (PCD) in tobacco BY-2 cells. We have recently shown that DHS triggers a production of H₂O₂, via the activation of NADPH oxidase(s). However, this production of H₂O₂ is not correlated with the DHS-induced cell death but would rather be associated with basal cell defense mechanisms. In the present study, we extend our current knowledge of the DHS signaling pathway, by demonstrating that DHS also promotes a production of nitric oxide (NO) in tobacco BY-2 cells. As for H₂O₂, this NO production is not necessary for cell death induction.Key words: tobacco BY-2 cells, sphingolipids, LCBs, dihydrosphingosine, sphinganine, apoptosis, programmed cell death (PCD), nitric oxide (NO)These last few years, it has been demonstrated in plants that long chain bases (LCBs), the sphingolipid precursors, are important regulators of different cellular processes including programmed cell death (PCD).^1–3 Indeed, plant treatment with fumonisin B1 or AAL toxin, two mycotoxins that disrupt sphingolipid metabolism, leads to an accumulation of the dihydrosphingosine (d18:0, DHS), one of the most abundant free LCB in plants and correlatively to the induction of cell death symptoms.^4,5 A more recent study shows a rapid and sustained increase of phytosphingosine (t18:0), due to a de novo synthesis from DHS, when Arabidopsis thaliana leaves are inoculated with the avirulent strain Pseudomonas syringae pv. tomato (avrRpm1), known to induce a localized PCD called hypersensitive response (HR).⁶ More direct evidences were obtained from experiments on Arabidopsis cells where external application of 100 µM C2-ceramide, a non-natural acylated LCB, induced PCD in a calcium (Ca²⁺)-dependent manner.⁷ Recently, we have shown that DHS elicited rapid Ca²⁺ increases both in the cytosol and the nucleus of tobacco BY-2 cells and correlatively induced apoptotic-like response. Interestingly, blocking nuclear Ca²⁺ changes without affecting the cytosolic Ca²⁺ increases prevented DHS-induced PCD.⁸Besides calcium ions, reactive oxygen species (ROS) have also been suggested to play an important role in the control of PCD induced by sphingolipids in plants.⁹ Thus, the C2-ceramide-induced PCD in Arabidopsis is preceded by an increase in H₂O₂.⁷ However, inhibition of ROS production by catalase, a ROS-scavenging enzyme, did not prevent C2-ceramide-induced cell death, suggesting that this PCD is independent of ROS generation. Moreover, we recently showed in tobacco BY-2 cells that DHS triggers a dose-dependent production of H₂O₂ via activation of a NADPH oxidase.¹⁰ The DHS-induced cytosolic Ca²⁺ transient is required for this H₂O₂ production while the nuclear calcium variation is not necessary. In agreement with the results of Townley et al. blocking the ROS production using diphenyleniodonium (DPI), a known inhibitor of NADPH oxidases, does not prevent DHS-induced cell death. Gene expression analysis of defense-related genes, using real-time quantitative PCR (RT-qPCR) experiments, rather indicates that H₂O₂ generation is likely associated with basal defense mechanisms.¹⁰In the present study, we further investigated the DHS signaling cascade leading to cell death in tobacco BY-2 cells, by evaluating the involvement of another key signaling molecule i.e., nitric oxide (NO). In plants, NO is known to play important roles in numerous physiological processes including germination, root growth, stomatal closing and adapative response to biotic and abiotic stresses (reviewed in ref. ^11–14). NO has also been shown to be implicated in the induction of PCD in animal cells,¹⁵ in yeast,¹⁶ as well as in plant cells, in which it is required for tracheid differentiation¹⁷ or HR activation.^18,19 Interestingly in the latter case, the balance between NO and H₂O₂ production appears to be crucial to induce cell death.²⁰ Here we show in tobacco BY-2 cells that although DHS elicits a production of NO, this production is not necessary for the induction of PCD.     

Why have chloroplasts developed a unique motility system?

Organelle movement in plants is dependent on actin filaments with most of the organelles being transported along the actin cables by class XI myosins. Although chloroplast movement is also actin filament-dependent, a potential role of myosin motors in this process is poorly understood. Interestingly, chloroplasts can move in any direction and change the direction within short time periods, suggesting that chloroplasts use the newly formed actin filaments rather than preexisting actin cables. Furthermore, the data on myosin gene knockouts and knockdowns in Arabidopsis and tobacco do not support myosins'' XI role in chloroplast movement. Our recent studies revealed that chloroplast movement and positioning are mediated by the short actin filaments localized at chloroplast periphery (cp-actin filaments) rather than cytoplasmic actin cables. The accumulation of cp-actin filaments depends on kinesin-like proteins, KAC1 and KAC2, as well as on a chloroplast outer membrane protein CHUP1. We propose that plants evolved a myosin XI-independent mechanism of the actin-based chloroplast movement that is distinct from the mechanism used by other organelles.Key words: actin, Arabidopsis, blue light, kinesin, myosin, organelle movement, phototropinOrganelle movement and positioning are pivotal aspects of the intracellular dynamics in most eukaryotes. Although plants are sessile organisms, their organelles are quickly repositioned in response to fluctuating environmental conditions and certain endogenous signals. By and large, plant organelle movements and positioning are dependent on actin filaments, although microtubules play certain accessory roles in organelle dynamics.^1,2 Actin inhibitors effectively retard the movements of mitochondria,^3–6 peroxisomes,^5,7–11 Golgi stacks,^12,13 endoplasmic reticulum (ER),^14,15 and nuclei.^16–18 These organelles are co-aligned and associated with actin filaments.^{5,7,8,10–12,15,18} Recent progress in this field started to reveal the molecular motility system responsible for the organelle transport in plants.¹⁹Chloroplast movement is among the most fascinating models of organelle movement in plants because it is precisely controlled by ambient light conditions.^20,21 Weak light induces chloroplast accumulation response so that chloroplasts can capture photosynthetic light efficiently (Fig. 1A). Strong light induces chloroplast avoidance response to escape from photodamage (Fig. 1B).²² The blue light-induced chloroplast movement is mediated by the blue light receptor phototropin (phot). In some cryptogam plants, the red light-induced chloroplast movement is regulated by a chimeric phytochrome/phototropin photoreceptor neochrome.^23–25 In a model plant Arabidopsis, phot1 and phot2 function redundantly to regulate the accumulation response,²⁶ whereas phot2 alone is essential for the avoidance response.^27,28 Several additional factors regulating chloroplast movement were identified by analyses of Arabidopsis mutants deficient in chloroplast photorelocation.^29–32 In particular, identification of CHUP1 (chloroplast unusual positioning 1) revealed the connection between chloroplasts and actin filaments at the molecular level.²⁹ CHUP1 is a chloroplast outer membrane protein capable of interacting with F-actin, G-actin and profilin in vitro.^29,33,34 The chup1 mutant plants are defective in both the chloroplast movement and chloroplast anchorage to the plasma membrane,^22,29,33 suggesting that CHUP1 plays an important role in linking chloroplasts to the plasma membrane through the actin filaments. However, how chloroplasts move using the actin filaments and whether chloroplast movement utilizes the actin-based motility system similar to other organelle movements remained to be determined.Open in a separate windowFigure 1Schematic distribution patterns of chloroplasts in a palisade cell under different light conditions, weak (A) and strong (B) lights. Shown as a side view of mid-part of the cell and a top view with three different levels (i.e., top, middle and bottom of the cell). The cell was irradiated from the leaf surface shown as arrows. Weak light induces chloroplast accumulation response (A) and strong light induces the avoidance response (B).Here, we review the recent findings pointing to existence of a novel actin-based mechanisms for chloroplast movement and discuss the differences between the mechanism responsible for movement of chloroplasts and other organelles.     

Cell Migration from Baby to Mother

Fetal cells migrate into the mother during pregnancy. Fetomaternal transfer probably occurs in all pregnancies and in humans the fetal cells can persist for decades. Microchimeric fetal cells are found in various maternal tissues and organs including blood, bone marrow, skin and liver. In mice, fetal cells have also been found in the brain. The fetal cells also appear to target sites of injury. Fetomaternal microchimerism may have important implications for the immune status of women, influencing autoimmunity and tolerance to transplants. Further understanding of the ability of fetal cells to cross both the placental and blood-brain barriers, to migrate into diverse tissues, and to differentiate into multiple cell types may also advance strategies for intravenous transplantation of stem cells for cytotherapeutic repair. Here we discuss hypotheses for how fetal cells cross the placental and blood-brain barriers and the persistence and distribution of fetal cells in the mother.Key Words: fetomaternal microchimerism, stem cells, progenitor cells, placental barrier, blood-brain barrier, adhesion, migrationMicrochimerism is the presence of a small population of genetically distinct and separately derived cells within an individual. This commonly occurs following transfusion or transplantation.^1–3 Microchimerism can also occur between mother and fetus. Small numbers of cells traffic across the placenta during pregnancy. This exchange occurs both from the fetus to the mother (fetomaternal)^4–7 and from the mother to the fetus.^8–10 Similar exchange may also occur between monochorionic twins in utero.^11–13 There is increasing evidence that fetomaternal microchimerism persists lifelong in many child-bearing women.^7,14 The significance of fetomaternal microchimerism remains unclear. It could be that fetomaternal microchimerism is an epiphenomenon of pregnancy. Alternatively, it could be a mechanism by which the fetus ensures maternal fitness in order to enhance its own chances of survival. In either case, the occurrence of pregnancy-acquired microchimerism in women may have implications for graft survival and autoimmunity. More detailed understanding of the biology of microchimeric fetal cells may also advance progress towards cytotherapeutic repair via intravenous transplantation of stem or progenitor cells.Trophoblasts were the first zygote-derived cell type found to cross into the mother. In 1893, Schmorl reported the appearance of trophoblasts in the maternal pulmonary vasculature.¹⁵ Later, trophoblasts were also observed in the maternal circulation.^16–20 Subsequently various other fetal cell types derived from fetal blood were also found in the maternal circulation.^21,22 These fetal cell types included lymphocytes,²³ erythroblasts or nucleated red blood cells,^24,25 haematopoietic progenitors^7,26,27 and putative mesenchymal progenitors.^14,28 While it has been suggested that small numbers of fetal cells traffic across the placenta in every human pregnancy,^29–31 trophoblast release does not appear to occur in all pregnancies.³² Likewise, in mice, fetal cells have also been reported in maternal blood.^33,34 In the mouse, fetomaternal transfer also appears to occur during all pregnancies.³⁵     

Roles of calcineurin B-like protein-interacting protein kinases in innate immunity in rice

Cytosolic free Ca²⁺ mobilization induced by microbe/pathogen-asssociated molecular patterns (MAMPs/PAMPs) plays key roles in plant innate immunity. However, components involved in Ca²⁺ signaling pathways still remain to be identified and possible involvement of the CBL (calcineurin B-like proteins)-CIPK (CBL-interacting protein kinases) system in biotic defense signaling have yet to be clarified. Recently we identified two CIPKs, OsCIPK14 and OsCIPK15, which are rapidly induced by MAMPs, involved in various MAMP-induced immune responses including defense-related gene expression, phytoalexin biosynthesis and hypersensitive cell death. MAMP-induced production of reactive oxygen species as well as cell browning were also suppressed in OsCIPK14/15-RNAi transgenic cell lines. Possible molecular mechanisms and physiological functions of the CIPKs in plant innate immunity are discussed.Key words: PAMPs/MAMPs, calcium signaling, CBL-CIPK, hypersensitive cell death, reactive oxygen speciesCa²⁺ plays an essential role as an intracellular second messenger in plants as well as in animals. Several families of Ca²⁺ sensor proteins have been identified in higher plants, which decode spatiotemporal patterns of intracellular Ca²⁺ concentration.^1,2 Calcineurin B-Like Proteins (CBLs) comprise a family of Ca²⁺ sensor proteins similar to both the regulatory β-subunit of calcineurin and neuronal Ca²⁺ sensors of animals.^3,4 Unlike calcineurin B that regulates protein phosphatases, CBLs specifically target a family of protein kinases referred to as CIPKs (CBL-Interacting Protein Kinases).⁵ The CBL-CIPK system has been shown to be involved in a wide range of signaling pathways, including abiotic stress responses such as drought and salt, plant hormone responses and K+ channel regulation.^6,7Following the recognition of pathogenic signals, plant cells initiate the activation of a widespread signal transduction network that trigger inducible defense responses, including the production of reactive oxygen species (ROS), biosynthesis of phytoalexins, expression of pathogenesis-related (PR) genes and reorganization of cytoskeletons and the vacuole,⁸ followed by a form of programmed cell death known as hypersensitive response (HR).^9,10 Because complexed spatiotemporal patterns of cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) have been suggested to play pivotal roles in defense signaling,^1,9 multiple Ca²⁺ sensor proteins and their effectors should function in defense signaling pathways. Although possible involvement of some calmodulin isoforms^11–13 and the calmodulin-domain/calcium-dependent protein kinases (CDPKs)^14–19 has been suggested, other Ca²⁺-regulated signaling components still remain to be identified. No CBLs or CIPKs had so far been implicated as signaling components in innate immunity.