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Cell-autonomous viral defense involving oxidative burst?

Cell-autonomous viral defense involving oxidative burst?


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I'm looking for a mechanism by which a cell detects a virus (probably a retrovirus) within itself, then triggers an oxidative burst in response. This should all happen within the cell itself, independent of any immune-specific cells outside. Ultimately interested in humans, but any pointers whatsoever would help.


So, the cell may trigger an oxidative burst in response to pathogen (not specifically virus).

In case of retroviruses, they are hard to detect for the cell (example HIV) as they use the cell machinery for replication and can also stay in inactive latent state thereby not triggering immune response.

Hope you found your answer. Thank you and good day.


RNA silencing suppression by plant pathogens: defence, counter-defence and counter-counter-defence

Coevolution between plants and pathogens has given rise to a large variety of defence pathways and counter-acting virulence pathways. Plant resistance against bacterial and fungal pathogens is largely mediated by a first layer of basal resistance and a second layer of strong race-specific resistance successful pathogens have evolved effector molecules to suppress both layers of defence. Analogously, virus resistance is mediated broadly by RNA silencing and also by race-specific resistance.

Viruses actively suppress RNA silencing, which alleviates anti-viral defence but also disrupts endogenous host gene regulation through microRNAs and small-interfering RNAs. Virulent infections thus cause disease partly through disrupting host silencing, while viruses unable to suppress silencing fail to replicate in healthy plants.

RNA silencing actively contributes to defence against bacterial and eukaryotic pathogens as well, through regulation of host gene expression. These non-viral pathogens have consequently evolved silencing suppressors that contribute to disease.

Recent work has revealed a crucial role for multiple aspects of host silencing in repressing race-specific defence responses. Thus, resistance and defence-promoting genes are silenced by miRNAs, siRNAs and/or epigenetically through DNA methylation.

Silencing suppressors deployed by pathogens therefore prevent the first layer of defence responses, but relieve repression of strong race-specific resistance mechanisms, which can contribute to increased host resistance.

This review synthesizes the intricate interplay between plant resistance pathways, RNA silencing and its manipulation by pathogens.


INTRODUCTION

Plant metabolism must be highly regulated in order to allow effective integration of a diverse spectrum of biosynthetic pathways that are reductive in nature. This regulation does not completely avoid photodynamic or reductive activation of molecular oxygen to produce reactive oxygen species (ROS), particularly superoxide, H2O2 and singlet oxygen ( Halliwell 1981 Fridovich 1998 ). However, in many cases, the production of ROS is genetically programmed, induced during the course of development and by environmental fluctuations, and has complex downstream effects on both primary and secondary metabolism. Plant cells produce ROS, particularly superoxide and H2O2, as second messengers in many processes associated with plant growth and development (e.g. Schroeder, Kwak & Allen 2001a Schroeder et al. 2001b Foreman et al. 2003 ). Moreover, one of the major ways in which plants transmit information concerning changes in the environment is via the production of bursts of superoxide at the plasma membrane ( Doke et al. 1994 ). Situations which provoke enhanced ROS production have in the past been categorized under the heading of ‘oxidative stress’, which in itself is a negative term implying a harmful process, when in fact it is probably in many cases quite the opposite, enhanced oxidation being an essential component of the repertoire of signals that plants use to make appropriate adjustments of gene expression and cell structure in response to environmental and developmental cues. Rather than involving simple signalling cassettes, emerging concepts suggest that the relationship between metabolism and redox state is complex and subtle.

Numerous redox components can be sensed by the cell to effect acclimatory changes that are mediated at diverse levels of regulation, and it is becoming apparent that redox modulation of protein function is a much more widespread phenomenon than previously considered. The extensive literature on ROS and antioxidants has previously been reviewed by ourselves ( Noctor & Foyer 1998 Foyer & Noctor 2000, 2003 ) and others ( Dat et al. 2000 Mittler 2002 Mahalingam & Federoff 2003 Conklin & Barth 2004 Mittler et al. 2004 Baier et al. 2005 ). Since it is not our intention to reiterate this information, we will concentrate on how plants might use and sense ROS and the two major redox buffers, ascorbate and glutathione (GSH) to regulate gene expression and plant function.


Introduction

Mammalian cells encode numerous pattern recognition receptors (PRRs) that sense invading pathogens and initiate innate immune responses through cytokine and chemokine production [1]. With viral pathogens, the type I interferon (IFN) family of cytokines serves as a first line of defense and is essential for controlling virus replication and pathogenesis. The IFN-induced antiviral response results from the transcription of hundreds of interferon-stimulated genes (ISGs), many of which inhibit different steps of the viral life cycle [2, 3]. Although less studied, the type I IFN response is also induced by many bacterial pathogens including Legionella pneumophila, Helicobacter pylori, Francisella tularensis, Yersinia pseudotuberculosis, Mycobacterium tuberculosis, and Listeria monocytogenes [4]. However, the role of type I IFN in bacterial infection remains unclear and systematic studies to uncover the breadth of ISGs targeting a bacterial pathogen have not been carried out.

We chose to clarify these aspects of IFN biology by using Listeria monocytogenes (herein referred to as Lm) as a model pathogen as its cellular life cycle has been described in detail and it exhibits a complex relationship with the mammalian IFN response system [5]. Lm is a Gram-positive food-borne pathogen that causes severe and life threatening disease in immunocompromised individuals, pregnant women, elderly and children [6]. Upon invasion of enterocytes, hepatocytes, or phagocytes, Lm gains access to the cytoplasm by lysing the primary phagosome. Lm rapidly replicates in the cytoplasm and spreads to adjacent cells via actin-based protrusion machinery [7]. Recent studies show that Lm stimulates the type I IFN response by secreting cyclic diadenosine monophosphate (c-di-AMP) that activates the Stimulator of Interferon Genes (STING). Activation of STING results in IRF3 phosphorylation and transcription of IFN genes [8, 9]. Notably, STING-deficient mice fail to produce IFNβ in response to Lm infection [10].

While the relationship between IFN and in vivo Lm infection has been firmly established, some discrepancies do exist between these studies. Early work showed that IFNβ increases the tolerance of mice to intravenous systemic Lm infection [11]. Similarly, Ifnar1 is required for resistance of mice to Lm invasion through the intestinal tract, further demonstrating a protective effect of IFN for a natural route of infection [12]. However, more recent studies indicate that mice lacking a functional type I IFN receptor (Ifnar1-/-) display greater resistance to intravenous Lm infection, suggesting that IFN exacerbates systemic Lm infection [13–15]. The type I IFN response has also been found to suppress adaptive immunity against Lm, since Sting-deficient mice exhibit greater numbers of cytotoxic lymphocytes and show protection from Lm reinfection after immunization [16]. These various effects of type I IFN on Lm infection likely reflect the different routes of Lm infection and the pleiotropic roles of IFN in distinct tissue environments or cellular populations encountered by the pathogen. Nevertheless, it is clear that type I IFN plays a significant role in shaping the host-pathogen interaction in vivo.

Because IFN induces a robust transcriptional response, the regulatory role of IFN in bacterial infection likely depends on the cellular expression of ISGs. However, the functions of most ISGs in immunity have not yet been elucidated due to the technical challenges of studying complex transcriptional responses at single-gene resolution. Recently, overexpression screens have been designed to study individual ISG functions [17–20]. While these approaches have proven to be highly successful for identifying genes that potently suppress invasion, replication, or egress of a wide variety of viruses, similar screening methodologies have not yet been adapted for bacterial pathogens. Here, we performed a gain-of-function screen of over 350 human type I IFN ISGs to identify genes that regulate Lm infection. This screen revealed potent bacterial restriction factors including MYD88, UNC93B1, TRIM14, AQP9, and MAP3K14. We demonstrated that the signaling adaptor MYD88 restricts Lm infection through the stimulation of a robust host gene expression program. In contrast, TRIM14 inhibited Lm infection through a non-transcriptional mechanism, thus suggesting that IFN stimulates diverse antibacterial properties. Importantly, we identified the human high affinity immunoglobulin receptor FcγRIa (CD64) as an IgG-independent enhancer of Lm internalization and established its role in the entry of Lm into phagocytic cells. Taken together, these findings reveal effector molecules involved in the complex relationship between Lm and the IFN response system, and open new avenues for exploring the cell-autonomous immune regulation of other bacterial pathogens.


Prophylactic Measures in Fields

Because viral agents are obligate intracellular parasites, curative treatments of virus infections are impossible, making viral diseases very difficult to control in fields. Prophylactic control measures are therefore crucial in combating epidemics on crops. They consist mainly on combining cultural practices, biosecurity measures and organism-vector management (Figure 1).

FIGURE 1. Prophylactic measures and main crop improvement strategies employed to control plant viral diseases.

Perform Regular Inspection for the Presence of Viral Pathogens

In this domain, the rising up of molecular biology techniques combined to continuous characterization of new etiological agents improved significantly the sensitivity, the specificity and the rapidity required to an accurate diagnosis of plant pathogenic viruses (Boonham et al., 2014). The reliability of the available diagnosis tests is a key point in viral disease management in fields, as infected plants need to be eradicated as fast as possible to minimize the virus spread.

Monitor Organism-Vector Populations

Plant viruses need to be transmitted by an organism-vector (insects, nematodes, zoosporic endoparasites) for their plant-to-plant spread. Hence, viral diseases can be efficiently controlled by limiting the populations of their vectors with the applications of appropriate pesticides. The use of non-host “trap plants” may be also considered to attract vectors to reduce the number of individuals feeding on the crop of interest and thus, the transmission of the disease (Bragard et al., 2013).

Set Up a Rigorous Control Program on Weeds and Other Host Plants in the Vicinity of the Field

Epidemics often arise from new viruses or new variants of classic viruses that spilled over from reservoir species to crops. Although this phenomenon results from a complex evolutionary process in which the main players are ecological factors, virus genetic plasticity and host factors, viral diseases can be controlled by managing the spatial structure and composition of field parcels, which impacts resistance durability (Elena et al., 2011 Fabre et al., 2012).

Respect the Phytosanitary Measures Decreed by Various International Commissions

Minimizing viral epidemics involves the respect of international legislations concerning worldwide trade of virus-free plant material, which applies to any development stage of a plant that can be carrier of viruses (seeds or fruit stones, grafts, rootstocks, seedlings, flowers. ), as well as manipulation of decontaminated horticultural tools.

Use Crop Cultivars that are Resistant to Viruses

The use of genetically resistant plants is one of the most efficient, sustainable and frequently employed strategies to control virus infections in fields. For centuries, it has involved plants selected by breeders for their agronomic proprieties combined to the absence of disease symptoms. However, from the middle of the 20th century, plant improvement programs capitalize strongly on the knowledge associated to plant–virus interactions to develop resistant varieties exploitable in agriculture.


Acknowledgements

We thank Josephine Blaazer for assisting the B. cinerea assays, Lisong Ma and Hanna Richter for generating the transgenic tomato and Arabidopsis lines and Bart Thomma and Jan van Kan (WUR, the Netherlands) for providing the V. dahliae and B. cinerea strains. X.D. and L.C. were financially supported by the CSC program. FT received funding from the European Union's Horizon 2020 research and innovation program under the Marie Skodowska-Curie grant agreement no. 676480 (Bestpass) and from the NWO-Earth and Life Sciences funded VICI project no. 865.14.003. M.J.B. is supported by the BBSRC (UK, relevant grant: J004553) and by the John Innes Foundation. The co-ordinates and structure factors for Avr2 have been deposited at the PDB with accession number 5OD4.


INTRODUCTION

There is a great diversity of external signals to which cells must respond. Independent of the simplicity or complexity of the cell type, the biochemical and molecular mechanisms to which cells need to respond are universal. Throughout different developmental stages, cells are susceptible to a variety of external signals from both the environment and neighboring cells. Indeed, cells must be ready to respond to these signals, and they do so through different types of universal signal transduction mechanisms.

Plant cells are no different from the most complex eukaryotic cells. However, because of their sessile nature, plants must respond to drastic environmental changes to survive, and they must adjust their growth and developmental behaviors in response to daily and seasonal environmental changes in a timely manner. An intriguing and important question in our understanding of a plant's developmental programs and environmental responses involves the types of strategies and mechanisms that plant cells use for the transmission and integration of various developmental signals.

Phospholipids are a major and vital component of all biological membranes and play a key role in processes, such as signal transduction, cytoskeletal rearrangement, and membrane trafficking. Genetic studies using Arabidopsis thaliana confirm that changes in phospholipid homeostasis profoundly affect plant growth and development. For example, the over-accumulation of phosphatidylinositol-4,5-bisphosphate (PIP2) and inositol-1,4,5-triphosphate (IP3) is characteristic of sac9 mutants, which show a constitutive stress response ( 1 ).

The route of phosphoinositides is one of the most important in plant signaling, and as such, they are located in all cell membranes there is evidence to suggest that phosphatidylinositol-specific phospholipase C (PI-PLC) is one of the component in this pathway involved in stress responses. Furthermore, changes in phosphoinositide levels have been characterized in a number of different plant species, and the stimulation of this signaling pathway is involved in many different plant reactions to environmental factors, such as drought, cold, salinity, and pathogen attack ( 2 ). PIP2 turnover is stimulated by the drought hormone abscisic acid in the stomata of Vicia faba ( 3 ) and by osmotic stress in A. thaliana cell cultures ( 4 ).

Phospholipid metabolism is also affected by the metal aluminum (Al), with the most important physiological consequence of Al-toxicity being a cessation of root growth and changes in root morphology this suggests that the root cytoskeleton is a target structure. This article discusses aluminum toxicity in plants and the impact that this metal has on phospholipid cell membranes of important agronomical plants in Mexico, namely Coffea arabica.

Aluminum Toxicity in Plants

Aluminum (Al) is the most abundant metallic constituent in the crust of the earth only the elements oxygen and silicon, which are both nonmetals, are more abundant. Al is never found as a free metal but is commonly found as aluminum silicate or as a silicate of aluminum mixed with other metals, such as sodium, potassium, iron, calcium, or magnesium. These silicates are not useful ores because the process of extracting Al from them is chemically difficult and expensive. Bauxite, an impure hydrated aluminum oxide, is the commercial source of Al and its compounds ( 5 ).

Al-toxicity is an important growth-limiting factor for plants in many acidic soils with a pH below 5.0. However, this toxicity can occur at pH levels as high as 5.5 ( 6 , 7 ). This problem is particularly serious in strongly acidic subsoils that are difficult to lime ( 8 ), where the challenge has been intensified by ongoing heavy applications of acid-forming nitrogenous fertilizers. High subsoil-acidity (Al-toxicity) reduces plant-rooting depth, increases susceptibility to drought, and decreases the use of subsoil nutrients ( 5 ).

At low soil-pH levels, solubilized Al ions (mainly in the phytotoxic form of Al) severely inhibit root elongation. Thus, Al-toxicity is a serious problem that causes decreased plant growth in acidic soils around the world ( 9 ). Indications that Al interferes with signal transduction pathways in cells have been observed ( 10 ).

Plants showing symptoms of aluminum toxicity are more sensitive to changes in environmental conditions, and this sensitivity can be caused by aluminum-mediated effects on any number of signal transduction cascades. Al accumulation is localized primarily at the root apex, suggesting that Al interacts with actively dividing and expanding cells. Among Al-toxicity symptoms, the main responses include inhibition of root growth and induction of callose (β-1,3-glucan) synthesis after a short-term treatment with Al. Both events have been related to oxidative stress induced by Al treatment ( 11 ), but the mechanism of signaling in response to Al remains unclear. The cumulative data on Al interactions indicate that Al has a significant effect on different signal transduction pathways in plants, such as phosphoinositide ( 12 ) and protein phosphorylation pathways, and that anion channels may participate in these interactions by excreting organic acids as an Al-tolerance mechanism.

In most plant species, especially Al-sensitive and crop species, Al uptake is limited mainly to the root system, where it accumulates predominantly in the epidermis and the outer cortex ( 13 , 14 ). However, there are many plant species that accumulate considerable amounts of Al in their shoots ( 15 ). These plants, frequently called hyperaccumulators, are mainly woody plants from tropical or subtropical regions, such as some species native to the region of central Brazil. Tea plant (Camellia sinensis), hydrangea and members of the Rubiaceae family are classic examples of hyperaccumulator plants ( 14 ).

On the other hand, there is unfortunately not much information in the literature related to the mechanism, cellular localization, or chemical form of the Al that accumulates in these plants. In tea leaves, most Al is chelated to the catechin group of polyphenols and, to a lesser extent, to phenolic and organic acids ( 16 ). In hydrangea leaves, Al is found as a complex with citrate ( 17 ), and in the hyperaccumulator plant Melastoma malabathricum Al is found bound to oxalate.

Likewise, it has been suggested that the rapid inhibition of root growth by Al treatment indicates more rapid signal transduction processes may be involved in causing this response. In particular, special attention has been paid to the phosphoinositide-associated transduction pathway because early research with animal cells indicated that cellular mechanisms of Al-toxicity could involve interactions between Al and components of the pathway ( 18 ).

Yakimova et al. ( 19 ) evaluated the effects of aluminum on signal transduction involving phosphoinositides and their possible relationship with cell death in tomato plants. The results suggested that low concentrations of heavy metal ions stimulate both PLC and phospholipase D (PLD) signaling pathways, which lead to the production of reactive oxygen species (ROS) and subsequent cell death executed by caspase-like proteases. Thus, this study demonstrated that the phospholipid signaling pathway is considered to be one of the important plant-signaling mechanisms involved in cell death. However, it remains difficult to determine the signaling pathways and where and how plants accumulate Al to counteract its toxic effects if the mechanism for how cells sense the presence of this metal is unknown.

Genes Expressed in the Presence of Aluminum

In recent years, considerable evidence has emerged in the literature that Al promotes oxidative stress in plant cells, although certain conditions are required for this to occur. Whether or not Al-induced oxidative stress is a primary or secondary effect is still a matter of debate. However, lipid peroxidation has been frequently observed as an early symptom. The Al-induced genes encoding proteins that function to overcome oxidative stress (e.g., glutathione S-transferase, peroxidase, blue copper-binding protein, phenylalanine ammonia lyase, 1,3-β-glucanase, and cysteine proteinase) have been previously reported ( 18 , 20 ). In addition, it was shown that the expression of these Al-induced genes in transgenic Arabidopsis plants conferred Al-tolerance and enhanced oxidative stress ( 21 ).

The first gene controlling Al 3+ resistance in plants was isolated from wheat ( 22 ). The Triticum aestivum aluminum-activated malate transporter (TaALMT1) gene encodes a member of the aluminium activated malate transporter (ALMT) family that consists of membrane-bound proteins ( 23 ). TaALMT1 functions as an Al 3+ -activated anion channel, releasing malate from root cells ( 24 ). It has also been shown that several other members of the ALMT family contribute to Al 3+ resistance in cereal and noncereal species in a similar manner. These discoveries were exciting at the time because it appeared as though a single gene family controlled Al 3+ resistance in a diverse range of species ( 25 ).

However, the model soon required revision after major resistance genes in sorghum and barley were mapped and sequenced. Aluminum resistance in these species relies on citrate efflux, and the proteins involved are not ALMTs but members of a completely different family of proteins called the multidrug and toxic compound extrusion (MATE) family. The MATE family of transporter proteins is a large and diverse group present in both prokaryotic and eukaryotic cells. Many of these proteins appear to function as secondary carriers for the removal of small organic compounds from the cytosol ( 26 , 27 ).

Studies on the heterologous expression and homology of the ALMT1 and MATE genes, in addition to the exploitation of available information on the physiology and genetics of resistance in other species, have uncovered additional resistance genes ( 28 ). This approach has helped identify candidate resistance genes in Arabidopsis ( 29 ), Brassica napus ( 30 ), rye ( 31 ), wheat ( 32 ), sorghum ( 33 ), and maize ( 34 ).

Aluminum and Coffee

The Rubiaceae family contains 500 genera, but Coffea is by far the most commercially significant genus in the family. Over 100 species have been described within Coffea L., and at least 25 major species likely exist, all of which are indigenous to tropical Africa and certain islands in the Indian Ocean. All Coffea species are woody but range in size from small shrubs to large trees over 10 m tall. Despite this diversity, only two species, both from Africa, are grown commercially on a large scale as follows: Coffea arabica L. (Arabica type coffee) and Coffea canephora Pierre ex Froechner (Canephora or Robusta type coffee) ( 35 , 36 ). C. arabica is a major crop worldwide and is the most heavily traded commodity apart from oil, accounting for 4% of the total world food trade. Coffee production has been negatively affected by factors such as disease and aluminum toxicity, the mitigation of which will require research into disease susceptibility, photosynthetic efficiency, water utilization, and tolerance to both soil acidity and Al content.

Coffee is often grown in acidic soils that have high Al 3+ levels. Al 3+ is an interchangeable form that can be released into the soil, making it very accessible to plants ( 37 ). Al 3+ also produces toxic effects in plants ( 38-40 ). Aluminum can be transferred to plants from the soil, which can modify regulation of the soil dynamics. Therefore, an understanding of the concentrations and dynamics of heavy metals in soils is necessary to determine the adequate properties and conditions needed for optimal agricultural working conditions ( 41 , 42 ).

Suspension Cells as a Model Tool

In vitro cell and tissue cultures from plants may provide an adequate system in which to perform studies on metal toxicity. An embryogenic-suspension cell line of Coffea arabica var. Catuai was obtained from a dispersed callus. The callus was originally obtained from the cotyledonary leaves of zygotic embryos from seeds cultured in vitro in Murashige–Skoog media at half the normal ionic strength and at a pH of 4.3. Under these conditions, the ability to grow in the presence of aluminum was diminished (Fig. 1) ( 43 ). Using this cell line as a model, we have focused on searching for the signaling pathway associated with growth inhibition that results from Al-mediated toxicity.

Cell suspensions of C. arabica were grown in Murashige–Skoog media at half the normal ionic strength, subcultured for 14 days, coated with gold particles and then observed under the scanning electron microscope. (A) and (B) control cells, (C) cells treated with 100 μM of AlCl3 for 30 min and (D) cells treated with 100 μM of AlCl3 for 1 h.

Al Effect on Phospholipid Signaling

In coffee cells, Al is known to produce growth inhibition at different concentrations. Al has also been related to different biochemical processes involving membrane phospholipids as well as several enzymes, such as PLC and PLD and the second messenger phosphatidic acid (PA). The breakdown of PIP2 into IP3 and diacylglycerol (DAG) by the action of PLC plays an important role in signal transduction pathways ( 44-47 ). IP3 and DAG produced by PIP2 hydrolysis act as second messengers, triggering the release of Ca 2+ from internal stores and activating protein kinases, respectively ( 45 ). In addition, PIP2 is important for the regulation of cytoskeletal dynamics, vesicle trafficking and ion transport a change in the phospholipid composition can noticeably affect cell function ( 46 ).

However, increasing experimental evidence has suggested that this pathway is not as simple as was first proposed. For example, levels of PIP2 are also regulated by different lipid kinases and lipid phosphatases. In addition, PIP2 regulates the activity of several enzymes and participates in the regulation of membrane trafficking. DAG can be a substrate for DAG kinase (DGK), in turn generating PA, and IP3 can also be phosphorylated by inositol kinases to generate IP6 ( 46 ), among others.

In previous studies focused on changes of lipids in response to Al-toxicity, Zhang et al. ( 48 ) found that when roots were treated with Al for 1 day, no effects were observed on the general lipid composition. In contrast, they also reported that Al treatment for 3 days induces modifications in the patterns of phospholipids, free sterols, fatty acids, and triacylglycerols. Other reports have shown that Al affects PLC activity ( 49 ) and interactions with enzymatic catalytic metal binding sites, specific membrane lipids, and ion channels ( 50 ).

Because of the suggestion that PLC is a main target for Al-toxicity, our group has been studying the effect of AlCl3 on the different components of this pathway, namely PLC and lipid kinases ( 51 ), using suspensions of C. arabica cells as a model ( 43 ). Two main effects were seen when cells were treated with AlCl3. In periods as short as 1 Min, Al-exposed cells displayed up to a twofold increase in their PLC and IP3 formation activities. Over longer periods, PLC activity was inhibited by more than 50%. It is important to note that this is the first report describing PLC activation by Al exposure. The activity of phosphatidylinositol 4-kinase (PI 4-K), phosphatidylinositol phosphate 5-kinase (PIP 5-K), and DGK increased when cells were incubated in the presence of different concentrations of AlCl3 ( 51 ). These results strongly support the theory that Al disrupts the metabolism of membrane phospholipids, regulating not only PLC, but also other enzymes that have key roles in signal transduction pathways.

PLD is ubiquitous in plants and hydrolyzes the terminal phosphodiester bond of phospholipids, in turn generating a free head group and PA. PA itself acts as a signaling molecule and is the precursor of additional regulatory molecules, including DAG pyrophosphate, lyso-PA, and arachidonic acid ( 46 ).

We prelabeled cells with 32 Pi and assayed for 32 P–PA formation in response to Al 3+ . Treatment of the cells with either AlCl3 or Al(NO3)3 for 15 min inhibited the formation of PA. To test how Al 3+ affects PA signaling, we used the peptide mastoparan-7 (mas-7), which is known as a very potent stimulator of PA formation. Al 3+ inhibited the mas-7-mediated induction of PA levels, both before and after incubation with Al 3+ . The PA involved in signaling is generated by two distinct phospholipid signaling pathways: via PLD or PLC and via DGK ( 52 ). By labeling with 32 Pi for short periods of time, we found that PA formation was inhibited by almost 30% when the cells were incubated with AlCl3, suggesting involvement in the PLC/DGK pathway. The effects of incubating the cells with the PLC inhibitor U73122 on PA formation were similar to those seen in response to AlCl3. In vivo PLD activation by mas-7 was reduced by Al 3+ . These results suggest that PA formation is prevented through the inhibition of PLC activity, and these data provide the first evidence for the effects of Al-toxicity on PA production ( 52 ).

While the existence of a plant receptor specific for Al 3+ has not been demonstrated, enhanced expression of a cell wall-associated kinase receptor upon Al treatment has been found ( 53 ). Perhaps the signal transduction cascade(s) activated by Al and the regulation of different pools of PA may be reflected in Al-toxicity as well as in Al tolerance. Whether or not the PA that forms by the two different routes has the same composition of fatty acids and the same target within the cell remains unknown.

Aluminum and the Protein Phosphorylation Signaling Pathway

Protein phosphorylation plays an important role in the regulation of various biological processes in plants ( 54 , 55 ). Protein phosphorylation has also been demonstrated to provide a signal transduction pathway for mediating extracellular stimuli in cells ( 56 ).

The aspect of Al-toxicity and signal transduction that has been most intensely studied to date involves phosphoinositide turnover, but protein phosphorylation is known to be one of the post-transduction modifications that regulates physiological events at the cellular level. Genetic, biochemical, and pharmacological analyses have shown that protein kinases and protein phosphatases play important roles in environmental stress responses. Studies examining the effect of Al on protein phosphorylation were not available until the last decade. In 2001, two reports examining the role of protein phosphorylation in Al-toxicity were published ( 57 , 58 ).

Suspension cells of C. arabica were incubated with increasing concentrations of AlCl3 (200–1,000 μmol L −1 ), and an in vitro phosphorylation reaction with cell extracts was performed. No changes in the proteins present in extracts from cells treated with AlCl3 were detected as compared with the proteins in extracts from untreated cells. However, the protein phosphorylation patterns did change. Phosphorylated proteins with molecular masses of 18, 31, and 53 kDa increased dramatically after in vivo treatment of cells with AlCl3. When AlCl3 was added to the reaction mixture, no differences in phosphorylation patterns were observed ( 57 ).

Although Al-induced organic acid exudation has been extensively documented [for a review, see ref. 38], signal transduction pathways that lead to this event are less well understood. Osawa and Matsumoto ( 58 ) have suggested that protein phosphorylation is required for signal transduction in Al-activated malate efflux from wheat root apex, likely through phosphorylation and subsequent activation of anion channels ( 58 ). In this study, treatment with the protein kinase broad-range inhibitor K-252a blocked Al-induced malate efflux at the root apex, and in-gel kinase assays with myelin basic protein, a substrate specific to mitogen-activated protein kinases (MAPK), showed that Al treatment induces the activation of a 48-kDa protein kinase. This putative MAPK was rapidly and transiently activated, and its activity increased from 0.5 to 5 min after Al exposure and subsequently diminished after 5 min. In addition, the activity of the 48-kDa kinase was approximately 10-fold higher after treatment with Al than without, and the Al-induced activation was lost within 5 min. The authors suggested that Al transiently activates this protein kinase quickly enough to precede the initiation of malate efflux ( 58 ). However, whether or not the 48 kDa protein kinase is directly involved in the pathway for malate efflux remains unknown. This kinase appears to play an essential role in transduction of the Al signal and expression of resistance mechanisms in the root apex of Al-resistant wheat ( 58 ).

Aluminum has been shown to be able to induce a rapid and transient activation of a putative 58 kDa MAPK protein in coffee suspension cells ( 59 ). Although this cell suspension culture showed a basal level of malate exudation ( 60 ), no direct evidence suggested that activation of the 58 kDa MAPK-like protein is related to Al resistance in coffee. The oxidative burst evoked by Al may directly affect MAPK signaling cascades ( 61 ). In Arabidopsis, H2O2 activates the MAPKs MPK3 and MPK6 via the MAP kinase kinase kinase Arabidopsis NPK1 (Nicotiana protein kinase 1)-like protein kinase 1 (ANP1) ( 62 ). Indeed, overexpression of the ANP1 gene in transgenic plants resulted in increased tolerance to cold, heat shock, and salinity ( 62 ). H2O2 also increases expression of the nucleotide diphosphate (NDP) kinase 2 in Arabidopsis ( 63 ). The overexpression of AtNDPK2 reduced H2O2 accumulation and enhanced tolerance to multiple stresses, including cold, salt, and oxidative stress. The effects of NDPK2 may be mediated by the MAPKs MPK3 and MPK6, as NDPK2 can interact with and activate the MAPKs. Therefore, these studies clearly suggest that abiotic stresses (including those from Al) induce ROS generation, which in turn activates MAPK signaling pathways.

ROS can also affect other signaling pathways. Kawano et al. ( 64 ) have observed that Al-induced ROS activates Ca 2+ influx at the plasma membrane, leading to an increase in the cytosolic Ca 2+ concentration. Briefly, the authors reported on aluminum-induced O2 •– accumulation via membrane-bound nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (optimal [AlCl3] = 6 mM), and, following an acute spike of O2 •– , a gradual increase in the cytosolic free Ca 2+ concentration ([Ca 2+ ]c) was detected. This increase in [Ca 2+ ]c was exclusively a consequence of Al-induced ROS production. However, the Al concentration that was optimal for O2 •– was inhibitory for [Ca 2+ ]c, implying that high concentrations of Al are inhibitory not only to cation channels but also to the H2O2-induced influx of Ca 2+ ( 64 ). These data also suggest that aluminum may induce Ca 2+ -dependent signaling events at the beginning of treatment, but a gradual increase in Al n+ activity in cells eventually turns off this signal. An increased cytosolic Ca 2+ concentration could activate other proteins such as PLD. PA is a second messenger that enhances the activity level of proteins, including protein kinases and phosphoenolpyruvate carboxylase ( 46 , 65 ). Because PA is a well-known activator of MAPK cascades in response to a variety of environmental stresses ( 65 ), a possible relationship between PA levels and MAPK regulation by Al may contribute to the Al-resistance response, as proposed in a recent report ( 52 ).

RNA Interference and its Potential to Elucidate Signaling Networks

Increased attention has been given to RNAi (RNA interference) as a potential therapeutic agent in the treatment of various diseases in humans ( 66 ). To date, the results of its application for the treatment of diseases such as cancer and viral infections ( 66 , 67 ) and those that affect the eye ( 68 ), nervous system ( 69 ), and bones[0] ( 70 ) have been significant and substantial. However, one limitation of the application of RNAi as a therapeutic agent in any disease is the strategy for liberation of the small molecules of siRNA (small interfering RNA). In this respect, changes have been made to these small molecules, ranging from the alteration of the phosphate backbone of the siRNA to the use of a complex involving lipids ( 71 ).

Much of the progress in the application of RNAi has been achieved in animal cells, and this knowledge has also been applied to plants. RNAi in plants is primarily used to determine the role of genes involved in different metabolic pathways with the aim of improving nutrient content and reducing the production of toxins ( 72 ).

Because plants are sessile organisms, they must find ways to detect and respond to external stimuli and to convert these stimuli into internal signals. As mentioned earlier, one of the first signaling pathways involved in converting a vast majority of environmental stimuli into signaling pathways is the phosphoinositide pathway. Some of the enzymes involved in this pathway have been observed to be regulated by many environmental factors, including changes in osmolarity, salinity ( 73 ), oxidative stress, metals, and pathogens ( 74 ).

Two common methods for the characterization of genes include the selection of mutants with desired phenotypes and the insertion of a transgene into the chromosome through genetic transformation. Although valuable, these methods are very time-consuming. However, the application of RNAi as a tool for assessing gene function using cell suspensions or protoplasts in combination with transient transformation assays can specifically facilitate the silencing of a large number of genes in a short period of time ( 75 ).

A protoplast is a plant cell that has been completely removed from the cell wall using mechanical and/or enzymatic approaches. In 1960, Cocking was the first to isolate protoplasts from higher plants by enzymatic methods. Using this method, it is currently possible to easily obtain protoplasts of whole organs and plant tissues in culture. These cellular systems are currently used as tools in concert with approaches, such as mutagenesis, selection, genetic transformation or fusion, somatic hybridization ( 76 ), assessment of gene function by RNAi, and the introduction of foreign DNA to study protein localization, ion channels, transport processes, cell division, and morphogenesis. Therefore, protoplasts can be used as a cellular model for RNAi experiments to answer the following questions regarding the phosphoinositide signal transduction pathway:What is the effect of transient PLC silencing on the activity of other phospholipases?

Does a protein signal transduction pathway regulate phospholipids in response to the RNAi-mediated silencing of PLC?

What happens to the activity of lipid kinases, such as PI4-K and PIP5-K, after the silencing of PLC or the silencing of the entire family?

RNAi has been used in greater depth to study phosphoinositide signaling in animals and has demonstrated that some enzymes, such as the lipid kinase phosphatidylinositol 3-kinase (PI3-K), are involved in the proliferation of cancer cells. However, in plants, interest in the study of phosphoinositides has been generated as a result of the enzymatic components and second messengers that are involved in processes, such as vesicular trafficking and pathogen attack, among others. Enzymes such as PLD and myo-inositol-1-phosphate synthase have been silenced in plants (including tomato and soybean) to determine their functions ( 77 , 78 ).

Therefore, we propose the use of RNAi to evaluate the function of each of the enzymes in the phosphoinositide signal transduction pathway and the levels of their product, PA, in response to different stimuli by silencing one, two or all of the enzymes in this pathway.

Model for the Effects of Al on the Different Signal Transduction Pathways

The possible effects of Al on signal transduction pathways are represented in Fig. 2. The effects of Al could occur in one of two ways as follows: (1) Al could interact directly with a receptor (R) on the membrane surface, or with the membrane itself, to initiate a secondary messenger cascade that then regulates the activity of different enzymes, such as PLC, PLD, lipids, or protein kinases, followed by a consequent activation of an anion channel or (2) Al enters into the cytoplasm and regulates the different enzymes as mentioned earlier, affecting anion channels directly or indirectly via secondary messengers. The regulation (up or down) of second messenger production may lead to protein phosphorylation through the activation of protein kinases, such as MAP kinases, cyclin dependent kinases, or others that may affect transcription factors and gene expression. Further studies will be required to corroborate this model and improve the understanding of aluminum-affected metabolic events in plants, specifically to elucidate the elements that constitute the Ca 2+ activated MAPK cascade and the role of phospholipid signaling in Al resistance in plants.

Model of the Al 3+ effect on PA formation. Al 3+ inhibits PLC and decreases DAG availability, reducing the amount of DAG phosphorylation to PA. This inhibition affects PA signaling levels by impairing the response of PA target proteins to signal cascades. The PA formed by PLD could lead a signaling cascade for Al 3+ toxicity tolerance by releasing organic acids through anionic channels activated by phosphorylation that involves MAPK signaling.


Perspectives

A role for DDR pathways leading to disease tolerance and inflammation modulation is perhaps not surprising in evolutionary terms, as many pathogens have been documented to induce DNA damage either as a necessary step or as collateral damage of their life cycle [5] . In addition, reactive oxygen and nitrogen species are produced by macrophages and neutrophils at the sites of infection as part of the resistance strategy [105] . We therefore propose that during evolution, DDR mechanisms have co-opted pathways dedicated to deal with stress, particularly, the initiation of an immune response and the subsequent termination of the inflammatory and repair responses. The molecular mechanisms that link DDR, aging, inflammation, metabolism, and disease tolerance remain very poorly defined. An additional problem is the often conflicting role of DNA damage in inflammation and modulation of cell death or survival. These are no doubt context and dose dependent, but the determination of the underlying switches and mechanisms of their modulation by DDR pathways will be of the highest interest and relevance. A critical need and opportunity therefore exist in this field, not only because progress in this area will lead to considerable novel insights into the pathophysiology of organ and organismal function but also because the molecular pathways involved are likely to constitute attractive new therapeutic targets for multiple conditions ranging from critical illness to aging.


Evaluation of the probe 2',7'-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress

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Conclusions

Oxidative stress is a powerful stimulator of eryptosis and contributes to the development of anemia in a variety of clinical conditions. The stimulation of eryptosis may further compromise microcirculation, as eryptotic erythrocytes adhere to the vascular wall. In view of the profound impact of anemia and impaired microcirculation, treatment of affected patients with eryptosis-inhibiting substances, such as several antioxidants, may be desirable. The possibility, however, must be kept in mind that the disruption of eryptosis may enhance hemolysis of defective erythrocytes with subsequent renal injury. Further in vivo studies are, thus, required to elucidate the beneficial and/or untoward effects of antieryptotic treatment in counteracting anemia associated with a wide range of systemic diseases.



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