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.     

Nitric oxide functions in both methyl jasmonate signaling and abscisic acid signaling in Arabidopsis guard cells

Intracellular components in methyl jasmonate (MeJA) signaling remain largely unknown, to compare those in well-understood abscisic acid (ABA) signaling. We have reported that nitric oxide (NO) is a signaling component in MeJA-induced stomatal closure, as well as ABA-induced stomatal closure in the previous study. To gain further information about the role of NO in the guard cell signaling, NO production was examined in an ABA- and MeJA-insensitive Arabidopsis mutant, rcn1. Neither MeJA nor ABA induced NO production in rcn1 guard cells. Our data suggest that NO functions downstream of the branch point of MeJA and ABA signaling in Arabidopsis guard cells.Key words: abscisic acid, Arabidopsis thaliana, guard cells, methyl jasmonate, nitric oxideStomatal pores that are formed by pairs of guard cells respond to various environmental stimuli including plant hormones. Some signal components commonly function in MeJA- and ABA-induced stomatal closing signals,¹ such as cytosolic alkalization, ROS generation and cytosolic free calcium ion elevation. Recently, we demonstrated that NO functions in MeJA signaling, as well as ABA signaling in guard cells.²NO production by nitric oxide synthase (NOS) and nitrate reductase (NR) plays important roles in physiological processes in plants.^3,4 It has been shown that NO functions downstream of ROS production in ABA signaling in guard cells.⁵ NO mediates elevation of cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt), inactivation of inward-rectifying K⁺ channels and activation of S-type anion channels,⁶ which are known to be key factors in MeJA- and ABA-induced stomatal closure.^2,7–9It has been reported that ROS was not induced by MeJA and ABA in the MeJA- and ABA-insensitive mutant, rcn1 in which the regulatory subunit A of protein phosphatase 2A, RCN1, is impaired.^7,10 We examined NO production induced by MeJA and ABA in rcn1 guard cells (Fig. 1). NO production by MeJA and ABA was impaired in rcn1 mutant (p = 0.87 and 0.25 for MeJA and ABA, respectively) in contrast to wild type. On the other hand, the NO donor, SNP induced stomatal closure both in wild type and rcn1 mutant (data not shown). These results are consistent with our previous results, i.e., NO is involved in both MeJA- and ABA-induced stomatal closure and functions downstream of the branching point of MeJA and ABA signaling in Arabidopsis guard cells.⁷ Our finding implies that protein phosphatase 2A might positively regulate NO levels in guard cells (Fig. 2).Open in a separate windowFigure 1Impairment of MeJA- and ABA-induced NO production in rcn1 guard cells. (A) Effects of MeJA (n = 10) and ABA (n = 9) on NO production in wild-type guard cells. (B) Effects of MeJA (n = 7) and ABA (n = 7) on NO production in rcn1 guard cells. The vertical scale represents the percentage of diaminofluorescein-2 diacetate (DAF-2 DA) fluorescent levels when fluorescent intensities of MeJA- or ABA-treated cells are normalized to control value taken as 100% for each experiment. Each datum was obtained from at least 30 guard cells. Error bars represent standard errors. Significance of differences between data sets was assessed by Student''s t-test analysis in this paper. We regarded differences at the level of p < 0.05 as significant.Open in a separate windowFigure 2A model of signal interaction in MeJA-induced and ABA-induced stomatal closure. Neither MeJA nor ABA induces ROS production, NO production, I_Kin and stomatal closure in rcn1 mutant. These results suggest that NO functions downstream of the branch point of MeJA signaling and ABA signaling in Arabidopsis guard cells.     

Step by Step: Deciphering Ion Transport in the Root Xylem Parenchyma

Proton pumps produce electrical potential differences and differences in pH across the plasma membrane of cells which drive secondary ion transport through sym- and antiporters. We used the patch-clamp technique to characterize an H⁺-pump in the xylem parenchyma of barley roots. This cell type is of special interest with respect to xylem loading. Since it has been an ongoing debate whether xylem loading is a passive or an active process, the functional characterization of the H⁺-pump is of major interest in the context of previous work on ion channels through which passive salt efflux into the xylem vessels could occur. Cell-type specific features like its Ca²⁺ dependence were determined, that are important to interpret its physiological role and eventually to model xylem loading. We conclude that the electrogenic pump in the xylem parenchyma does not participate directly in the transfer of KCl and KNO₃ to the xylem but, in combination with short-circuiting conductances, plays a crucial role in controlling xylem unloading and loading through modulation of the voltage difference across the plasma membrane. Here, our recent results on the H⁺ pump are put in a larger context and open questions are highlighted.Key Words: plant nutrition, H⁺-ATPase, anion conductance, K⁺ channel, electrophysiology, signaling networkThe root xylem parenchyma is of major interest with respect to nutrient (and signal) traffic between root and shoot. One of its main functions appears to be xylem loading. However, the cell walls of the vascular tissue provide apoplastic paths between xylem and phloem that represent the upward and downward traffic lanes, allowing nutrient circulation¹ (Fig. 1). Therefore mechanisms for ion uptake and for ion release must exist side by side. In the last 15 years major progress has been made in the investigation of transport properties of xylem-parenchyma cells, and both uptake and release channels and transporters were identified. Today, we have good knowledge on the role of K⁺ and anion conductances in xylem loading with salts.² Note, that from the functionally well characterized conductances only the molecular structure of K⁺ channels is known. In contrast, many transporters are identified on the molecular level, but functional data are scarce.Open in a separate windowFigure 1Distribution of tissues in the periphery of the stele. The stippled area marks the region from which early metaxylem protoplasts originated. E, Endodermis with Casparian strip; eMX, ‘early’ metaxylem vessel; IMX, ‘late’ metaxylem vessel; Mph, metaphloem (sieve tube); Pph, protophloem (sieve tube); P, pericycle; Cx, cortex. Symplasmic and apoplasmic transport routes are indicated in red and black, respectively. The Casparian strip prevents apoplastic transport into the stele. Plasmodesmata are shown exemplarily for the indicated symplastic pathway. All cells of the symplast are connected via plasmodesmata. Sites of active uptake into the root symplast and of release into the stelar apoplast are indicated by a black and an orange arrow. Modified from Wegner and Raschke, 1994.³A challenging question to deal with was the dispute about xylem loading with ions being a passive or active process. While it is clear that energy through electrogenic H⁺ efflux is needed to take up nutrient ions from the soil against their electrochemical gradient into the cortical symplast, it has been a matter of debate if ion release into xylem vessels also is energy-linked or if the electrochemical potentials of ions are raised high enough to allow a thermodynamically passive flux.^2,3 The Casparian strip prohibits apoplastic transport of nutrients into the stele and electrically insulates the stelar from the cortical apoplast. Therefore the electrical potential difference of the cells in the xylem parenchyma could be independent from the cortical potential difference but be subject to control, for instance, from the shoot.⁴ Indeed, evidence points to xylem loading as a second control point in nutrient transfer to the shoot.^5,6 The identification and characterization of K⁺ and anion conductances clearly showed that release of KCl and KNO₃ into the xylem can be passive through voltage-dependent ion channels.^2,3,7–9 No need appeared for a pump energizing the transfer of salts to the xylem.However, H⁺ pumps are ubiquitous. H⁺-ATPases are encoded by a multigene family and heterologous expression in yeast showed that isoforms have distinct enzymatic properties.^10,11 As the example of the amino acid transporter AAP6 from the xylem parenchyma shows, a cell-type specific functional characterization of transporters is essential to draw conclusions on their physiological role. AAP6 is the only member of a multigene family with an affinity for aspartate in the physiologically relevant range. The actual apoplastic concentration of amino acids and the pH will determine what is transported in vivo.^12,13 Xylem-parenchyma cells of barley roots were strongly labelled by antibodies against the plasma membrane H⁺-ATPase.¹⁴ In a recent publication in Physiologia Plantarum we report the functional analysis of the electrogenic pump from the plasma membrane of xylem parenchyma from barley roots that was done with the patch-clamp technique after specific isolation of protoplasts from this cell type. It displayed characteristics of an H⁺-ATPase: current-voltage relationships were characteristic for a ‘rheogenic’ pump¹⁵ and currents were stimulated by fusicoccin or by an enlarged transmembrane pH gradient and inhibited by dicyclohexylcarbodiimide (DCCD). Importantly, it also showed distinct characteristics. Neither intracellular pH nor the intracellular Ca²⁺ concentration affected its activity. Noteworthy, K⁺ and anion conductances from the same cell type are controlled by intracellular [Ca²⁺]^7,9 (Fig. 2). It was proposed that the effect of abscisic acid (ABA) on anion conductances is mediated via an increase in the cytosolic Ca²⁺ concentration.¹⁶ Very likely stelar H⁺ pumps are stimulated by ABA.¹⁷ Thus, a Ca²⁺ independent control has to be hypothesized in this case.Open in a separate windowFigure 2Control of ion conductances in the plasma membrane of xylem-parenchyma cells. Arrowheads indicate stimulation and bars indicate inhibition by an increase in cytosolic [Ca²⁺],^7,9,16 by ABA,^16,17,21 by cytosolic and apoplastic acidification,^4,22 by G-proteins²³ and by an increase in apoplastic [K⁺]⁷ and [NO₃⁻].²⁴ Apoplastic [K⁺] and [NO₃⁻] modify the voltage dependence exerting negative feedback on K⁺ efflux and a positive feedback on NO₃⁻ efflux. Abscisic acid has an immediate effect on ion channel activity, most likely via [Ca²⁺], and causes a change in gene expression as indicated by circles (up) and bars (down). ABA perception is not clear. A Ca²⁺ influx could occur through a hyperpolarization activated cation conductance (HACC).^16,25 Cation transporters are NORC, nonselective cation conductance, KORC, K⁺-selective outwardly rectifying conductance (=SKOR⁸), and KIRC, K⁺-selective inwardly rectifying conductance, and anion conductances with different voltage-dependencies and gating characteristics are X-QUAC, quickly activating anion conductance, X-SLAC, slowly activating anion conductance, and X-IRAC, inwardly rectifying anion channel.^2,3,9,16,26 Transported ions and direction of flux are plotted.To date, we know that besides Ca²⁺ and abscisic acid also the pH, nonhydrolyzable GTP analogs and extracellular NO₃⁻ and K⁺ affect membrane transport capacities of root xylem-parenchyma cells (Fig. 2). Other control mechanisms by metabolites, the redox potential and phytohormones have to be included, especially if they represent signals in xylem loading or root-shoot communication. The composition of the xylem sap changes during the course of a day, depending on nutrient supply and various stresses, and the apoplastic ion concentration is considered to be an important factor in ion circulation.^6,18,19 ABA is such a signal. It is known to increase solute accumulation within the root by inhibiting release of ions into the xylem.¹⁷ Any change in transport activity has an impact on the membrane potential. This again determines whether salt release or uptake takes place. Passive salt release is restricted to a limited range of membrane potentials in which conductances for anions and cations are active simultaneously, that is with depolarization. Negative membrane voltages will be required for reabsorption of NO₃⁻ by a putative NO₃⁻/H⁺-symporter and for the uptake of K⁺ and amino acids.^3,13 As shown in our recent paper, the balance between the activities of the H⁺-pump and the anion conductances could affect the position between a depolarized and a hyperpolarized state of the parenchymal membrane. Thus, H⁺ pump activity is crucial in membrane voltage control. Furthermore, the simultaneous activities of H⁺ pumps and anion conductances make the generation of a high pH gradient possible, whilst maintaining electroneutrality. The proton gradient could be used for ion transport through cotransporters and antiporters as suggested for the loading of borate into the xylem through the boron transporter BOR1.²⁰ So we are on the way to decipher xylem loading in roots and this exciting field will also provide information about small-scale nutrient cycling and root-shoot communication. To determine how the activities of pumps, channels and transporters are adjusted among each other is the next challenge. Further insight has to be obtained by experimentation as well as by biophysical modeling.     

Chemical Signal as a Rapid Long-Distance Information Messenger After Local Wounding of a Plant?

A series of works have described an important role of chemical signaling compounds in generation of the stress response of plants in both the wounded and distant undamaged plant tissues. However, pure chemical signals are often not considered in the fast (minutes) long-distance signaling (systemic response) because of their slow propagation speed. Physical signals (electrical and hydraulic) or a combination of the physical and chemical signals (hydraulic dispersal of solutes) have been proposed as possible linkers of the local wound and the rapid systemic response. We have recently demonstrated an evidence for involvement of chemical compounds (jasmonic and abscisic acids) in the rapid (within 1 hour) inhibition of photosynthetic rate and stomata conductance in distant undamaged tobacco leaves after local burning. The aim of this addendum is to discuss plausible mechanisms of a rapid long-distance chemical signaling and the putative interactions between the physical and chemical signals leading to the fast systemic response.Key Words: tobacco, local burning, systemic response, hydraulic surge, electrical signal, abscisic acid, jasmonic acidPlants have evolved an amazingly complex system of defence-related strategies to protect themselves upon local wounding.^1–7 Important characteristics of self-defence responses of plants are their velocity and ubiquity. Indeed, fast (minutes to hours) responses to injurious factors have been detected in the site of injury and in distant regions (systemic response) in various plants.^8–11 These findings suggest that a signal generated by an attack to one leaf is transmitted through a whole plant. Several kinds of chemical^3,6 and physical¹² signals induced by local wounding and even their combination¹³ have been implicated. However, a little is known about the interactions of these signals and about the mechanisms of initiation of the short-term systemic responses.We have used a model system—tobacco plants exposed to the local burning—to study the signals involved in rapid wound responses of photosynthetic apparatus.¹⁴ Local burning of an upper leaf of a tobacco plant induced rapid changes in surface electrical potential (within seconds) and a pronounced fast decline in the stomatal conductance, CO₂ assimilation and transpiration (within minutes) in the basipetal direction (Fig. 1). Moreover, we have detected a fast (within minutes) transient increase in levels of endogenous abscisic acid (ABA) followed by a huge rapid rise in endogenous jasmonic acid (JA) in the leaf below the burned one. ABA and jasmonates are known to be involved in signaling pathways leading to stomatal closure and downregulation of photosynthesis.^15,16 Increases in ABA and/or JA levels have only previously been detected in remote untreated tissues several hours after local wounding^8,9 suggesting that chemical signals are too slow to induce rapid systemic response. Previous works have reported that fast physical (electrical) signals play an essential role in short-term systemic photosynthetic responses.^11,17 However, a several-minutes delayed stomata closing response after the initiation of electrical potential changes has been reported in Mimosa¹⁸ and in our case in tobacco¹⁴ plants. Therefore, the guard cell deflation is most likely triggered not only by the electrical signal, but also by indirect factors. Based on close correlations, our results now provide a new evidence for the idea that chemical signals (ABA and mainly JA) participate in mediating the short-term systemic photosynthetic responses to local burning in tobacco plants.Open in a separate windowFigure 1The model of putative signalling pathways leading to the rapid systemic responses of tobacco plants to local burning. Hypothetical (dashed lines) local responses, generation of signals and transport processes and detected (full line) systemic responses are demonstrated. For details see the text.The question is how do the physical (electrical and/or hydraulic) and chemical signals act? They may independently induce specific elements of systemic responses. However, they are more likely to act in a coordinated, interactive fashion. In this scenario (see Fig. 1), within first minutes after the local burning, hydraulic surge transmitted basipetally and acropetally through the xylem would transport chemicals released at the wound site (hydraulic dispersal¹⁹) and evoke changes in the ion fluxes in surrounding living cells leading to the local electrical activity.^12,13 The hypothesis of hydraulic dispersal is supported by our preliminary experiments with the fluorescent dye Rhodamine B applied on cut petiole of the upper leaf of tobacco plants showing that solutes can be rapidly transported (within minutes) basipetally following wounding.The rapid kinetics and transient character of ABA accumulation¹⁴ suggest that the main transport mechanism is the hydraulic dispersal in xylem. The participation of ABA in the generation of systemic electrical activity and/or vice versa cannot be ruled out.^8,20A rapid hydraulically driven transport of chemicals in the xylem of wounded plant in a reversed (basipetal) direction^19,21 to transpiration stream is not generally accepted. Exposing of leaves of undamaged plants to radioactive labelled molecules to determine the speed of chemical signal transport could be misrepresent, because hydraulic signal is not generated in undamaged plants and then the detected transport speed is too slow. Moreover, previous work²² demonstrated that neither the mass flow itself, nor the associated pressure changes induce the systemic response (the proteinase inhibitor activity). Thus, the efficacy of chemical agents in rapid systemic signaling seems to depend on transport by the mass flows associated with hydraulic signals.¹⁹However, hydraulic dispersal acts only for minutes, until all water released at the wound site is exhausted.²¹ A requirement for hydraulic dispersal of any solute is its presence in the wounded tissue at the time of wounding.¹⁹ Detected slower kinetics of JA accumulation than in the case of ABA and the huge rise of JA levels¹⁴ indicate a systemic accumulation of JA also by some additional processes.Does additional JA accumulation result from de-novo synthesis in undamaged leaves as a response to physical signal or does it result from a JA transport from the wounded leaves? In the longer time-frame the phloem transport²³ should also be considered. Experiments with tomato plants have shown that de novo JA synthesis in distant leaves is not required for the systemic response and that biosynthesis of JA at the wound site is necessary for the generation of a systemic signal.⁷ Indeed, a short-term increase in endogenous concentrations of JA has been detected in wounded tissue in Nicotiana sylvestris⁹ and rice.¹⁰However, a rapid burst in the systemic JA accumulation found in our experiments¹⁴ would implicate an ultra-rapid and extreme JA accumulation at the wound site before its transport. The systemic JA accumulation (within 1 hour¹⁴) preceded the generation of enzymes involved in the JA biosynthesis in the wounded leaf.Thus, several processes are suggested to play a role in the ultra-rapid and huge JA accumulation:

1. initiation of JA accumulation by preexisting enzymes,²⁴
2. fast release of free JA from its storage pools in cells (e.g., JA-conjugates²⁵),
3. direct uptake of elicitors (JA) by the phloem of the wounded leaf and exchange between the xylem and phloem as a consequence of severe wounds,²⁶
4. the mass flow (containing remaining JA) driven mainly acropetally in the xylem by transpiration after damping the hydraulic surge,²¹
5. JA accumulation evoked by the fast transmitting physical (electrical or hydraulic) signal that leads to imbalances in ion fluxes,^8,12,27
6. JA accumulation (and subsequent transport) directly in the phloem, where JA biosynthetic enzymes are located (at least in tomato²⁴),
7. volatile chemical compounds (methylester of JA) spreading in the surrounding air of wounded leaf could serve as signaling molecules and sources of JA.^25,28

The relevance of the above mentioned mechanisms should be checked by further research. Complex quantitative and kinetic analysis of JA and ABA content, levels of its biosynthetic derivatives (also volatiles in the surrounding air) and simultaneous physical signal detection in wounded and distant unwounded tissues would fill the remaining void about their role and interactions in the wound signal transduction networks. In addition, a suppression of other signaling pathways with similar transport kinetics (e.g., volatile compounds transmission, systemin and oligosaccharides generation and/or transport, using mutant plants) would be useful.Substantial similarity between the rapid physical (electrical) signaling in animal nervous system compared with the physical (electrical) signaling in plants has already been reported.^29,30 Interaction of chemical and electrical signals is the process well documented for post-synaptic events in animals. Our data now strengthen the role of chemical signals next to the role of physical signals in plants in the rapid systemic wound response; such a role of chemicals in plants was often underestimated up to now.     

Effect of abscisic acid on symbiotic nitrogen fixation activity in the root nodules of Lotus japonicus

The phytohormone abscisic acid (ABA) is known to be a negative regulator of legume root nodule formation. By screening Lotus japonicus seedlings for survival on an agar medium containing 70 µM ABA, we obtained mutants that not only showed increased root nodule number, but also enhanced nitrogen fixation. The mutant was designated enf1 (enhanced nitrogen fixation 1) and was confirmed to be monogenic and incompletely dominant.In long-term growth experiments with M. loti, although some yield parameters were the same for both enf1 and wild-type plants, both the dry weight and N content of 100 seeds and entire enf1 plants were significantly larger compared than those traits in wild-type seeds and plants. The augmentation of the weight and N content of the enf1 plants most likely reflects the increased N supplied by the additional enf1 nodules and the concomitant increase in N fixation activity.We determined that the endogenous ABA concentration and the sensitivity to ABA of enf1 were lower than that of wild-type seedlings. When wild-type plants were treated with abamine, a specific inhibitor of 9-cis-epoxycarotenoid dioxygenase (NCED), which results in reduced ABA content, the N fixation activity of abamine-treated plants was elevated to the same levels as enf1. We also determined that production of nitric oxide (NO) in enf1 nodules was decreased. We conclude that endogenous ABA concentration not only regulates nodulation, but also nitrogen fixation activity by decreasing NO production in nodules.Key words: Lotus japonicus, symbiotic nitrogen fixation, nitric oxide, ABA, root nodulePhytohormones are known to be important for regulating the number of nodules established on the root of legumes.¹ For example, ethylene is a well-known negative regulator of nodulation, influencing the earliest stages from the perception of Nod factor to the growth of infection threads.^2–4 In contrast, cytokinin is a positive regulator of nodulation. The cytokinin insensitive mutant hit1 (loss-of-function) of Lotus japonicus and the snf2 (gain-of-function) mutants of Medicago truncatula provide genetic evidence demonstrating that cytokinin plays a critical role in the activation of nodule primordia.^5–7Abscisic acid (ABA), added at concentrations that do not affect plant growth, also negatively regulates nodulation in some legumes.^8–11 Recently, Medicago truncatula overexpressing abi1-1, a gene that encodes a mutated protein phosphatase of the type IIC class derived from Arabidopsis and that suppresses the ABA signaling pathway,^12,13 was shown to exhibit ABA insensitivity as well as a hypernodulating phenotype.¹⁴In this study, we isolated a Lotus japonicus (Miyakojima MG20) mutant that showed an increased root nodule phenotype and a lowered sensitivity to ABA, and proceeded to carry out its characterization. This mutant, named enf1 (enhanced nitrogen fixation 1) exhibit enhanced symbiotic N fixation activity. Most legume N fixation activity mutants, such as ign1, sen1 and sst1, are Fix-.^15–17At first, to obtain ABA-insensitive or low-sensitive mutants of Lotus japonicus, we treated Miyakojima MG20 with EMS to induce base substitutions randomly in the genome. M3 seeds were sown on an agar-solidified medium containing 70 µM ABA, a concentration that inhibits the germination of wild-type MG20 seeds. M4 plants obtained by the screening were inoculated with rhizobia (Mesorhizobium loti MAFF303099), and the number of nodules per plant was counted 35 days after inoculation (DAI). Plant No. 12 not only formed more root nodules than did the wild-type MG20 plants, but surprisingly it also exhibited increased nitrogen fixation activity per plant. Both mutant phenotypes were stably inherited in the M4 and M5 generation. Back-crossing mutant No. 12 to wild-type MG20 yielded 153 F2 progeny from which a line that showed the highest N fixation activity and more nodules per plant was derived. This line was designated enf1 (enhanced nitrogen fixation 1).At 28 DAI, the number of nodules formed on enf1 roots was approximately 1.7 times greater than that of MG20, and the N fixation activity per enf1 plant was elevated 1.8 times over that of the wildtype plants. Because the N fixation activity per unit of enf1 nodule weight was also increased 1.7 times, we concluded that the increased N fixation activity was not solely due to the enhanced number of root nodules.The endogenous ABA concentration and the sensitivity to ABA of enf1 were lower than those of wild-type seedlings. ABA is believed to regulate early nodulation stages negatively by inhibiting Nod factor signaling, bacterial infection, and nodule initiation.^14,18 Elongated ITs were more common in enf1 root hairs at later stages of development (8–12 DAI). Furthermore, ITs were detected in nodule primordia more frequently in enf1 compared to MG20. These results suggest that the earliest stages of nodule development are not as strongly inhibited in enf1 as they are in wild-type MG20.Because enf1 had a low endogenous ABA concentration, we hypothesized that the decrease in ABA concentration caused the elevation of N fixation activity. To test this hypothesis, we treated wild-type plants at 28 DAI with 20 µM abamine, a specific inhibitor of ABA synthesis.¹⁹ After a three day-treatment period, acetylene reduction activity was measured. Such short treatment periods of abamine are not expected to induce new nodule development. Wild-type plants treated with abamine had a reduced endogenous ABA concentration in roots, to about one-fourth of the level of control plants. However, N fixation activity was elevated to about 170% over the non-treated controls (Fig. 1A and B). This result phenocopies enf1, which shows decreased endogenous ABA concentration as well as elevated N fixation activity. These results strongly suggest that the decrease in endogenous ABA concentration in enf1 was responsible for the increased levels of N fixation activity. Applying 0.5 µM ABA did not result in a further increase in N fixation activity even though the endogenous ABA concentrations are presumed to increase (Fig. 1A and B).Open in a separate windowFigure 1Effects of ABAconcentration on nitrogen fixation activity. M. loti-inoculated plants were grown for 28 days on vermiculite-filled pots supplied with B & D medium. Plant roots 28 DAI were treated with 0.5 µM ABA, 20 µM abamine, with both ABA and abamine, or were untreated (B & D medium control), respectively, for 3 days. (A) ARA per nodule weight. (B) ABA concentration in root. At least 15 plants were used in acetylene reduction assay. Four different plants were used for the measurement of ABA concentration and 3 repeats were performed. Error bars indicate the standard error, and the significance of differences between untreated control and treated values was determined by the two-tailed multiple t-test with Bonferroni correction following ANOVA (three comparisons in four groups), *p < 0.05, **p < 0.01.Nitric oxide (NO) is known as a strong inhibitor of N fixation activity,²⁰ as well as a signal component in ABA signaling pathway.^21,22 NO production in root nodules formed by enf1 21 DAI and 28 DAI was examined by using the fluorescent dye diaminofluorescein-FM (DAF-FM), a NO specific detector, and relative fluorescence unit (RFU) values were estimated. The RFU values of enf1 nodules 21 DAI were clearly decreased compared with that of MG20; this trend was more obvious at 28 DAI. Moreover, the effect of reduced ABA concentration caused by treatment with abamine on NO production was analyzed (Fig. 2). When nodules formed on the roots of 28-d-old plants were treated, the RFU value of the enf1 mutant was almost the same for (−) abamine and (+) abamine-treated, whereas, the RFU value of abamine-treated MG20 plants was significantly reduced compared to untreated MG20 (Fig. 2). These results strongly suggest that decreased production of NO caused by the low concentration of ABA in enf1 nodules was responsible for the increase in N fixation activity.Open in a separate windowFigure 2NO production in nodules. Quantification of nitric oxide in nodules that were treated with abamine. Nodules on the root of 28-day-old plants were treated with 20 µM abamine for 3 days. Relative fluorescent units (RFU) per nodule fresh weight at 515 nm, normalized against MG20 plants, are shown. The data represent the average ± standard error of 3 independent experiments derived from nodules of 6 to 8 plants. The significance of differences among the four groups was determined by the two-tailed multiple t-test with Bonferroni correction following ANOVA (six comparisons in four groups) and the different letters refer to significant differences at p < 0.01.Until now, the majority of symbiotic mutants that have been described represents loss of or defects in root nodule formation.^6,23,24 Many of these mutants induce nodules that are Fix-.^15–17 Although reports of mutants that show increased root nodule number^25–28 or spontaneous root nodule formation exist,^7,29 reports concerning mutations where N fixation activity is elevated without deleterious effects on plant growth and development are limited. One exception is the L. japonicus rdh1 mutant, which also exhibits a hypernodulation and enhanced nitrogen fixation phenotype.³⁰In this report, we have shown that mutating the ENF1 gene leads to an elevation of N fixation activity without accompanying adverse growth effects. In long-term growth experiments, some yield parameters were the same for both enf1 and wild-type plants, but both the dry weight and N content of 100 seeds and entire enf1 plants were significantly larger compared to those parameters in wild-type seeds and plants. These results strongly suggest that more nitrogen is fixed in the enf1 mutant than in wild-type plants. Therefore, this gene should be an important target for molecular breeding. We have determined that ENF1 gene is inherited in a monogenic and incompletely dominant manner. Our future work will identify the gene responsible for these positive growth effects.     

The role of rice phenolics efflux transporter in solubilizing apoplasmic iron

Iron (Fe) is an essential micronutrient for plants whose deficiency presents a major worldwide agricultural problem. Moreover, Fe is not easily available in neutral to alkaline soils, rendering plants deficient in Fe despite its abundance. Plants secrete phenolics, such as protocatechuic acid (PCA) and caffeic acid (CA), to take up and utilize apoplasmic precipitated Fe, but despite the rapid progress in understanding cellular and subcellular Fe transport, the molecular mechanisms of phenolics synthesis and secretion are not clear. Recently, we isolated and characterized a phenolics efflux transporter in rice by characterizing a mutant in which the amount of PCA and CA in the xylem sap was dramatically reduced, which we hence named phenolics efflux zero 1 (pez1). PEZ1 is a plasma membrane protein that transports PCA when expressed in Xenopus laevis oocytes, and characterization of PEZ1 knockdown and overexpressing plants revealed that it plays an essential role in solubilizing precipitated apoplasmic Fe. The identification of PEZ1 will increase our understanding of apoplasmic Fe solubilization as well as promote research on phenolics efflux mechanisms in different organisms.Key words: iron, Oryza sativa, phenolics transport, protocatechuic acid, xylem sapAlthough mineral soils contain over 6% iron (Fe),¹ it predominantly exists as Fe(III) chelates, and plants ultimately cannot absorb Fe under various physiological conditions such as high soil pH in alkaline soils.² Thus, plants growing in high-pH soils are not very efficient in developing and stabilizing chlorophyll, resulting in the yellowing of leaves, poor growth and reduced yield. Plants, however, have developed sophisticated mechanisms to take up the small amount of soluble Fe. Non-graminaceous plants release protons, secrete phenolics, reduce Fe(III), and finally, take up Fe²⁺.^3–5 Once Fe is solubilized, Fe(III) is reduced to Fe²⁺ by a membrane-bound Fe(III) reductase oxidase.⁶ Then Fe²⁺ is transported into the root by an iron-regulated transporter (IRT1). In contrast, graminaceous plants rely on an Fe(III) chelation system through the secretion of mugineic acid (MA) family phytosiderophores.^3,7,8 The MAs are secreted to the rhizosphere through TOM1 ⁹ and then they chelate Fe(III); the resulting Fe-MA complex is transported by the Yellow Stripe family transporters (OsYSL15 in the case of rice¹⁰). Rice plants also have the ability to take up Fe²⁺ through the OsIRT1 transporter.¹¹In plants, Fe uptake from the apoplasm is well documented at the molecular level, with the exception of phenolics synthesis and efflux. Phenolics, such as protocatechuic acid (PCA), are reported to chelate Fe(III) solubilization and reduce it to Fe²⁺ in vitro.¹² Moreover, removing the secreted phenolics in hydroponic culture solution triggers Fe deficiency responses in roots by inhibiting the solubilization and utilization of apoplasmic Fe.¹³ In this manner, phenolics play a major role in Fe solubilization, besides which PCA and other phenolics play a diverse role in biological systems, such as acting as antioxidants and free radical scavengers, and in nitric oxide synthase.^14–17 Phenolics are also converted to lignin and suberin through the action of peroxidases.² The activity of peroxidases, as well as the formation of lignin, decreases under Fe deficient conditions.^2,18 As suberin plays an important role in controlling the movement of solutes,¹⁹ the role of phenolics in controlling water and mineral transport cannot be overlooked. Thus, understanding the molecular mechanism of phenolics efflux transport is crucial for developing strategies to mitigate widespread Fe deficiency.PEZ1 was isolated in an effort to characterize T-DNA mutants for genes regulated by cadmium (Cd). PEZ1 belongs to the multidrug and toxic compound extrusion transporter family whose members transport small organic compounds.²⁰ The substrates of PEZ1 were identified by analyzing liquid chromatography/mass spectrometry data profiles of the xylem sap of pez1-1 and pez1-2 mutants. The data indicated that PEZ1 transports PCA and caffeic acid (CA). Furthermore, PEZ1 transported radiolabeled PCA when expressed in Xenopus laevis oocytes. PEZ1 localizes to the plasma membrane in rice root cells, as well as in rice root hairs and onion epidermal cells. The pez1-2 mutant accumulated more Fe in the roots, but not in the leaves, compared to wild-type (WT) plants; the differences were greater in the presence of Cd, while no difference was observed in the accumulation of other metals. No significant difference was observed in zinc, manganese (Mn), and copper concentration between WT and pez1-2, in both the roots and shoots, with or without Cd. Fe concentration in the xylem sap was lower than in the WT, while no difference was observed for xylem Cd and Mn. Significant differences in the localization of insoluble Fe were observed when leaf samples were stained with Perl''s solution to examine the localization of Fe. These results suggested a clear role of PEZ1 in solubilizing precipitated apoplasmic Fe.²¹Secretion of excess PCA strongly solubilizes Fe precipitated in the stele, leading to symptoms of Fe excess. The analysis of PEZ1 overexpression lines confirmed this hypothesis. PEZ1 overexpression lines accumulated higher amounts of Fe in roots and leaves owing to the high solubilization of precipitated apoplasmic stele Fe, and as a result, the growth of these lines was severely restricted. In contrast, PEZ1 overexpression lines grew better than the WT in calcareous soil, showing that in these lines, PCA-solubilized Fe is available under Fe-limiting conditions.The expression of PEZ1 is regulated by Cd, and both of the PEZ1 knockdown mutants accumulated higher Cd amounts in leaves and seeds when grown in soil, without compromising morphological or physiological characteristics, like the SPAD value, leaf dry weight, yield, and the concentration of other metals in seeds. Why pez1 accumulates Cd is not clear. PCA has a lower affinity for Cd compared to glutathione, and PEZ1 does not transport Cd.²¹ Cd is partly transported through the Fe uptake system in plants.^22–26 Thus, in pez1, Cd accumulation seems to be triggered by the upregulation of OsIRT1. OsIRT1 localization in the phloem, its substrate specificity, and increased expression in pez1 mutants suggests that Fe and Cd uptake and translocation in pez1 mutants could be enhanced through OsIRT1,¹¹ and that an increased Cd accumulation in pez1 mutants may be due to the increase in OsIRT activity in a decreased Fe environment in which Cd will have reduced competition. PEZ1 localizes to the stele in root cells. The localization of different genes involved in Fe transport is summarized in Figure 1.Open in a separate windowFigure 1Tissue-specific expression of Fe homeostasis-related genes in rice root.In short, phenolics secretion affects Fe acquisition in rice. Reduced secretion of PCA in the pez1-2 mutant impairs the solubilization of precipitated apoplasmic Fe in the stele, and thus, the low availability of Fe leads to the induction of OsIRT1. As PEZ1 and OsIRT1 co-localize in the stele, the PCA secretion may complement Fe²⁺ uptake by OsIRT1 and seems to be an integral part of the Fe²⁺ uptake system in rice (Fig. 2). In contrast, the increase in phenolics secretion in PEZ1-overexpressing plants increases the solubilization of apoplasmic Fe, and plants showed an increased tolerance to Fe deficiency in alkaline soils. The identification of PEZ1 is an important step that helps in better understanding the solubilization of apoplasmic Fe and will generate research on phenolics efflux mechanisms in other plants.Open in a separate windowFigure 2Model of Fe and Cd uptake mechanisms in rice xylem. P.M., plasma membrane.     

Hydrogen sulfide effects on stomatal apertures

Hydrogen sulfide (H₂S) has recently been reported to be a signaling molecule in plants. It has been well established that is has such roles in animals and it has been suggested that it is included into the group of gasotransmitters. We have recently shown that hydrogen sulfide causes stomatal opening in the model plant Arabidopsis thaliana. H₂S can be supplied to the plant tissues from donors such as sodium hydrosulfide (NaSH) or more recently from slow release H₂S donor molecules such as GYY4137. Both give similar effects, that is, they cause stomatal opening. Furthermore both H₂S donors reduced the accumulation of nitric oxide (NO) induced by abscisic acid (ABA) treatment of leaf tissues. Here similar work has been repeated in a crop plant, Capsicum anuum, and similar data has been obtained, suggesting that such effects of hydrogen sulfide on plants is not confined to model species.Key words: abscisic acid, GYY4137, hydrogen sulfide, nitric oxide, stomatal apertureThe effects of hydrogen sulfide on plants have been studied for many years, but it is only recently that it has been suggested that this gas is acting as a signaling molecule. In animals this has been well established^1,2 and it has been suggested that H₂S be grouped together with other gasotransmitters.^2,3 This group will also contain nitric oxide (NO) which as well as having established roles in animals is also known to cause stomatal closure in plants.^4,5 With this in mind, we previously investigated whether H₂S may also have an effect on stomatal closure, using a model organism Arabidopsis thaliana.⁶ The study used two different H₂S donors, sodium hydrosulfide (NaSH) and morpholin-4-ium 4 methoxyphenyl(morpholino) phosphinodithionate (GYY4137). The former will release H₂S in an instant burst which soon dissipates, which questions the wisdom of its use. GYY4137 on the other hand will release H₂S much more slowly and in a manner which is more likely to reflect physiological generation of H₂S.^7,8 Both donors caused stomatal that had previously been exposed to light to open even further. If leaf tissues were not light treated H₂S compounds once again caused stomata to open. Furthermore, H₂S treatment prevented stomatal closure caused by dark treatment. To investigate the possible mechanism of this effect, tissues were treated with the plant hormone abscisic acid (ABA) to initiate NO generation and then NO accumulation was measured in the absence and presence of H₂S donors using fluorescent probes and confocal microscopy.⁹ Both NaSH and GYY4137 caused a reduction in the accumulation of NO. This suggests that H₂S may be acting by a disruption of NO signaling, which results in the alteration of guard cell physiology.Others have reported different effects of H₂S on stomatal movements. Garcia-Mata and Lamattina¹⁰ found that both H₂S donors NaSH and GY4137 caused stomatal closure in different plant species including Vicia faba, Arabidopsis thaliana and Impatiens walleriana. Use of glibenclamide, which is an ABC transport inhibitor, reduced the effect. Cystathione γ lyase and L-Cys desulfhydrase are enzymes which may be responsible for H₂S synthesis and stomatal movements were also reduced by propargylglycine, an inhibitor of these enzymes. It was suggested therefore that H₂S helps to mediate ABA signaling pathway in guard cells. This paper was further discussed following its publication by Desikan.¹¹ However, this seems to be in conflict with the work we reported. This would not be the first time that there has been contradictory data when it comes to reporting stomatal movements, as ethylene has been shown to mediate auxin-induced opening¹² and to cause stomatal closure.¹³More recently it has been reported that stomatal conductance was increased by carbonyl sulfide (COS).¹⁴ The authors went on to suggest that this effect was mediated by H₂S which was produced from COS hydrolysis. This seems to support our original data. Therefore, here we report on the effects of both NaSH and GYY4137 on a different plant species and one which has relevance as an important crop, that is Capsicum anuum. GYY4137 was supplied as in our previous paper in reference ⁶ and ⁷. As can be seen in Figure 1A NaSH caused stomata to open further, even though the leaf tissue had been exposed to the light. Stomata were able to close, as ABA treatment demonstrated, therefore showing that the stomata were not defective. When the experiments were repeated with GYY4137 (Fig. 1B) and smaller but similar effect of the addition of the H₂S donor was seen. This would be expected as the release of H₂S from GYY4137 would be slower and more prolonged than from NaSH.^7,8 To investigate if NO accumulation is also effected in Capsicum when treated with H₂S donors, leaf tissue was treated with ABA to initiate NO generation and NO measured by the use of DAF2-DA as previously reported in references ⁶ and ⁹. Once again the presence of H₂S donors dramatically reduced the amount of NO that was measured following ABA treatment (Fig. 2). This once again suggests that H₂S is having an effect on NO metabolism which may account for the stomata aperture measurements. It has been suggested in animal systems that H₂S and NO react, resulting in the formation of nitrosothiols/nitrothiol-like species¹⁵ which could have signaling effects in their own right. NO in plants has been reported to lead to increased cGMP and/or increased nitrosylation of proteins,⁵ but if H₂S was removing the bioavailability of NO both mechanisms are likely to be reduced.Open in a separate windowFigure 1H₂S donors cause stomatal opening in Capsicum anuum. The leaves of analyzed from Capsicum anuum plants which were between 6 and 7 weeks old. Stomatal bioassays were performed as described previously by Desikan et al.⁹ Epidermal peels were incubated in MES-KCl buffer [10 mM 2-morpholino ethane sulfonic acid (MES), 5 mM KCl, 50 µM CaCl₂, pH 6.15] for 2.5 h exposed to the direct lightning (in 60–100 IE m⁻² s⁻¹) before the addition of various compounds. (A) Samples were sheltered from direct lighting and treated with ABA or NaHS for 2.5 h and left under the day light conditions before stomata apertures were analyzed. (B) Samples were sheltered from direct lighting and treated with ABA or GYY 4137 for next 2 h and left under the day light conditions before stomata apertures were analyzed. Apertures were measured using a light microscope and imaging camera with LEICA QWIN image processing and analysis software (Leica Microsystems and Imaging Solutions, Cambridge, UK). n = 40 stomatal apertures, ±SE. GYY4137 was synthesis as previously described in reference ⁷.Open in a separate windowFigure 2H₂S donors reduce NO accumulation in Capsicum anuum. Nitric oxide accumulation was estimated using the specific NO dye DAF2-DA (Calbiochem, Nottingham, UK), using the method described previously by Desikan et al.⁹ Epidermal fragments in MES-KCl buffer (10 mM MES, 5 mM KCl, 50 µM CaCl₂, pH 6.15) were exposed to the direct lightning for 2 h. After 2 h samples were loaded with 30 µM DAF2-DA for 15 min before washing with MES-KCl buffer; three times for 10 min. Fragments were subsequently incubated for a further 30 min in the presence of various compounds (as indicated below) before images were visualized using CLSM (excitation 488 nm, emission 515 nm; Nikon PCM2000, Kingston-upon-Thames, UK). Images acquired were analyzed using SCION IMAGE software (Scion, Frederick, MD, USA). (A) Control with no treatment; (B) ABA (50) treatment; (C) NaHS (100 µm) treatment alone; (D) ABA treatment in the presence of NaHS; (E) GYY4137 (100 µm) treatment alone; (F) ABA treatment in the presence of NaHS.NO metabolism is involved in a wide range of plant functions, including seed germination,¹⁶ floral development,¹⁷ root gravitropism¹⁸ and gene expression¹⁹ as well as controlling stomatal function.⁴ H₂S on the other hand may be present in or around plants for a variety to reasons. H₂S can be produced endogenously by for example by plastid located cysteine desulfhydrases,²⁰ or H₂S may come from the environment,²¹ including the soil and waters.²² This is further discussed in a recent review in reference ²³. Therefore future work should be focused on the interplay between H₂S from a variety of sources on the NO metabolism of a range of plant tissues. Not all affects of H₂S will be mediated by NO, with alterations of glutathione on H₂S treatment being reported for example.²⁴ But the full extent of the modulation of NO accumulation and signal by both exogenous and endogenous H₂S needs to be explored so the role of these gasotransmitters^2,3 in mediating hormone and stress responses in plants can be fully understood.