Electrophysiological approach to examine a putative cold- and menthol-sensitive channel in the liverwort Conocephalum conicum

The mechanism of cold perception by plants is still poorly understood. It was found that temperature drop evokes changes in the activity of ion pumps and channels, which leads to plasma membrane depolarization.^1,2 The nature of the primary step of its action (alteration in membrane composition,³ transient influx of Ca²⁺ etc.,²) has not been elicited yet. Our electrophysiological experiments conducted on the liverwort Conocephalum conicum showed that its cells respond not only to sudden cooling⁴ but also to menthol, generating depolarization of the plasma membrane and action potentials (APs). Similar results are well documented in mammals; cold or “cooling compounds” including menthol cause activation of thermosenstitive channel TRPM8 permeable to Ca²⁺ and generation of AP series.⁵ TRP receptors are detected, among others, in green and brown algae. Possible existence of TRPM8-like channel-receptor in Conocephalum conicum is discussed here.Key words: action potential, cold, liverwort, menthol, thermoreceptors, voltage transient     

R type anion channel: A multifunctional channel seeking its molecular identity

Plant genomes code for channels involved in the transport of cations, anions and uncharged molecules through membranes. Although the molecular identity of channels for cations and uncharged molecules has progressed rapidly in the recent years, the molecular identity of anion channels has lagged behind. Electrophysiological studies have identified S-type (slow) and R-type (rapid) anion channels. In this brief review, we summarize the proposed functions of the R-type anion channels which, like the S-type, were first characterized by electrophysiology over 20 years ago, but unlike the S-type, have still yet to be cloned. We show that the R-type channel can play multiple roles.Key words: R-type anion channel, nitrate, sulphate, guard cell, action potentialAnion channels play a central role in signal transduction, nutrient transport and cell turgor regulation.¹ By far, their function was particularly well investigated in the guard cells of stomata using a combination of electrophysiological, pharmacological and genetic tools. In this system, anion channel activation was shown to be one of the limiting steps in the loss of cell turgor leading to stomatal closure.² In algal cells, anion channels were shown to contribute to membrane excitability through the generation of action potential.^1,3With the burst of molecular biology in the nineties, the genes coding for plant ion channels started to be unveiled. The first channel gene to be cloned in plant was the shaker-like potassium channel identified in a yeast functional expression screen.^4,5 More than ten years later, TaALMT1 and AtCLCa were characterized as the first members of two important anion channel families.^6,7 This growing group of newly identified channels, accounting for electrophysiological activity described long ago, includes the MSLs anion selective mechanosensitive channels.⁸ Recently, the well known S-type channel has been finally recognized to be encoded by members of the SLAC1 (and other SLAH) family (Slow Anion Channel-Associated 1).⁹ In agreement with electrophysiological data,^10–13 it requires phosphorylation by a Protein Kinase in order to be functional.^14,15 In contrast, the molecular identity of the R-type anion channel remains unknown. Therefore, this candidate, which has been functionally known since twenty years, remains the next challenge for plant channel physiologists.     

Historical Overview on Plant Neurobiology

The review tracks the history of electrical long-distance signals from the first recordings of action potentials (APs) in sensitive Dionea and Mimosa plants at the end of the 19^th century to their re-discovery in common plants in the 1950''s, from the first intracellular recordings of APs in giant algal cells to the identification of the ionic mechanisms by voltage-clamp experiments. An important aspect is the comparison of plant and animal signals and the resulting theoretical implications that accompany the field from the first assignment of the term “action potential” to plants to recent discussions of terms like plant neurobiology.Key Words: action potentials, slow wave potentials, plant nerves, plant neurobiology, electrical signaling in plants and animailsFor a long time plants were thought to be living organisms whose limited ability to move and respond was appropriately matched by limited abilities of sensing.¹ Exceptions were made for plants with rapid and purposeful movements such as Mimosa pudica (also called the sensitive plant), Drosera (sundews), Dionea muscipula (flytraps) and tendrils of climbing plants. These sensitive plants attracted the attention of outstanding pioneer researchers like Pfeffer,^2,3 Burdon-Sanderson,^4,5 Darwin,⁶ Haberlandt^7–9 and Bose.^10–13 They found them not only to be equipped with various mechanoreceptors exceeding the sensitivity of a human finger but also to trigger action potentials (APs) that implemented these movements.The larger field of experimental electrophysiology started with Luigi Galvani''s discovery of “animal electricity” or contractions of isolated frog legs suspended between copper hooks and the iron grit of his balcony.¹⁴ It soon became clear that the role of the electric current was not to provide the energy for the contraction but to simulate a stimulus that existed naturally in the form of directionally transmitted electrical potentials. Studies by both Matteucci and Du Bois-Reymond¹⁵ recognized that wounding of nerve strands generated the appearance of a large voltage difference between the wounded (internal) and intact (external) site of nerves. This wound or injury potential was the first, crude measurement of what later became known as membrane or resting potential of nerve cells. It was also found that various stimuli reduced the size of the potential (in modern terms: they caused a depolarization), and to describe the propagating phenomenon novel terms such as action potential (AP) and action current were created (reviewed in refs. ¹⁵ and ¹⁶). Rather than relying on such indirect methods, the membrane theory of exicitation proposed by Bernstein in 1912¹⁷ made it desirable to directly measure the value of cell membrane potentials. Such progress soon became possible by the introduction of microelectrodes (KCl-filled glass micropipettes with a tip diameter small enough to be inserted into living cells) to record intracellular, i.e., the real membrane potentials (V_m). The new technique was simultaneously adopted for giant cells (axons) of cephalopods such as Loligo and Sepia¹⁸ and giant internodal cells of Charophytic green algae. In the 1930s Umrath and Osterhout^19–21 not only made the first reliable, intracellular measurements of membrane potentials in plant cells (reporting V_m values between −100 to −170 mV) but the first intracellular recordings of plant APs as well. When this technique was complemented with precise electronic amplifiers and voltage clamp circuits in the 1940s, one could measure ion currents (instead of voltages) and so directly monitor the activity of ion channels. The smart application of these methods led to a new, highly detailed understanding of the ionic species and mechanisms involved in V_m changes, especially APs.^22–27 Whereas the depolarizing spike in animal nerve cells is driven by an increased influx of Na⁺ ions, plant APs were found to involve influx of Ca²⁺ and/or efflux of Cl⁻¹ ions.The first extracellular recording of a plant AP was initiated by Charles Darwin and performed on leaves of the Venus flytrap (Dionea muscipula Ellis) by the animal physiologist Burdon-Sanderson in 1873.^4–6 Ever since APs have often been considered to fulfil comparable roles in plants and nerve-muscle preparations of animals. However, this was never a generally accepted view. While it is commonly assumed that the AP causes the trap closure, this had not been definitely shown (see refs. ²⁸ and ²⁹). Kunkel (1878) and Bose (1907, 1926) measured action spikes also in Mimosa plants where they preceded the visible folding movements of the leaflets.^{12–13,30–31} Dutrochet and Pfeffer^2–3 had already found before that interrupting vascular bundles by incision prevented the excitation from propagating beyond the cut and concluded that the stimulus must move through the vascular bundles, in particular the woody or hadrome part (in modern terms the xylem). Haberlandt⁷ cut or steam-killed the external, nonwoody part of the vascular bundles and concluded that the phloem strands were the path for the excitation, a notion which is confirmed by a majority of recent studies in Mimosa and other plant species. APs have their largest amplitude near and in the phloem and there again in the sieve cells.^{23–24,32–35} Moreover, APs can be recorded through the excised stylets of aphids known to be inserted in sieve tube elements.^36–37 Other studies found that AP-like signals propagate with equal rate and amplitude through all cells of the vascular bundle.³⁸ Starting studies with isolated vascular bundles (e.g., from the fern Adiantum), Bose found increasing amplitudes of heat-induced spikes by repeated stimulation (tetanisation) and incubation in 0.5 % solution of sodium carbonate.^10–13 Since the electrical behavior of isolated vascular strands was comparable to that of isolated frog nerves, Bose felt justified to refer to them as plant nerves.Although at the time a hardly noticed event, the discovery that normal plants such as pumpkins had propagating APs just as the esoteric “sensitive” plants was a scientific breakthrough with important consequences.^39–40,32 First, it corrected the long-held belief that normal plants are simply less sensitive and responsive than the so-called “sensitive plants” from Mimosa to Venus flytraps. Second, it led to the stimulating belief that so widely distributed electric signals must carry important messages.⁴¹ The ensuing studies made considerable progress in linking electrical signals with respiration and photosynthesis,^40–42 pollination,^43–44 phloem transport^{33,36–37,45} and the rapid, plant-wide deployment of plant defenses.^46–53The detailed visualization of nerve cells with silver salts by the Spanish zoologist S. Ramon y Cajal, the demonstrated existence of APs in Dionea and Mimosa as well as the discovery of plant mechanoreceptors in these and other plants⁹ at the end of the century was sufficient stimulation to start a search for structures that could facilitate the rapid propagation of these and other excitation signals. Researchers began to investigate easily stainable intracellular plasma strands that run across the lumen of many plant cells, and sometimes even continue over several cells for their potential role as nerve-like, excitation-conducting structures. Such strands were shown to occur in traumatized areas of many roots⁵⁴ and in insectivorous butterworts where they connect the glue-containing hair tips with the basal peptidase-producing glands of the Pinguicula leaves.^55–56 However, after investigating these claims, Haberlandt came to the conclusion that the only nerve-like structures of plants were situated the long phloem cells of the vascular bundles.^7–8 From that time on papers, lectures and textbooks reiterated statements that “plants have no nerves”.This unproductive expression ignores the work of Darwin, Haberlandt, Pfeffer and Bose together with the fact that in spite of their anatomical differences, nerve cell networks and vascular bundles share the analog function of conducting electrical signals. Similar anatomical differences have not been an obstacle to stating that both plants and animals consist of cells. The mechanistic similarity of excitations (consisting of a transient decline in cell input resistance) in plant and nerve cells was later elegantly demonstrated by the direct comparison of action potentials in Nitella and the giant axon of squids.^57–58 Today, consideration of nerve-like structures in plants involves increasingly more aspects of comparison. We know that many plants can efficiently produce electric signals in the form of action potentials and slow wave potentials (= variation potentials) and that the long-distance propagation of these signals proceeds in the vascular bundles. We also know that plants like Dionea can propagate APs with high efficiency and speed without the use of vascular bundles, probably because their cells are electrically coupled through plasmodesmata. Other analogies with neurobiology include vesicle-operated intercellular clefts in axial root tissues (the so-called plant synapses)⁵⁹ as well as the certain existence and operation of substances like neurotransmitters and synaptotagmins in plant cells (e.g., refs. ⁶⁰ and ⁶¹). The identification of the role(s) of these substances in plants will have important implications. Altogether, modern plant neurobiology might emerge as a coherent science.⁶²Electrophysiological and other studies of long-distance signals in plants and animals greatly contributed to our knowledge of the living world by revealing important similarities and crucial differences between plants and animals in an area that might directly relate to their different capacities to respond to environmental signals. Even at this stage the results are surprising. Rather than lacking electric signals, higher plants have developed more than just one signal type that is able to cover large distances. In addition to APs that occur also in animals and lower plants,⁶³ higher plants feature an additional, unique, hydraulically propagated type of electric signals called slow wave potentials.⁶⁴     

Intracellular linkers are involved in Mg2+-dependent modulation of the Eag potassium channel

Modulation of activation kinetics by divalent ions is one of the characteristic features of Eag channels. Here, we report that Mg²⁺-dependent deceleration of Eag channel activation is significantly attenuated by a G297E mutation, which exhibits a gain-of-function phenotype in Drosophila by suppressing the effect of shaker mutation on behavior and neuronal excitability. The G297 residue is located in the intracellular linker of transmembrane segments S2 and S3, and is thus not involved in direct binding of Mg²⁺ ions. Moreover, mutation of the only positively charged residue in the other intracellular linker between S4 and S5 also results in a dramatic reduction of Mg²⁺-dependent modulation of Eag activation kinetics. Collectively, the two mutations in eag eliminate or even paradoxically reverse the effect of Mg²⁺ on channel activation and inactivation kinetics. Together, these results suggest an important role of the intracellular linker regions in gating processes of Eag channels.Key words: Drosophila potassium channel, activation, magnesium, gating, gain-of-function mutantThe Drosophila ether-à-go-go channel (Eag) is the founding member of an evolutionarily conserved subfamily of voltage-gated K⁺ channels,^1–5 which includes HERG (human eag-related gene), a channel that plays an important role in regulating cardiac excitability and maintaining normal cardiac rhythm.⁶ As demonstrated by previous studies from many groups, this family of channels is critical to the function of a variety of biological processes, including memory formation,⁷ signal transduction,^8,9 cell proliferation and tumor progression.^10–12In flies, mutation of eag increases neuronal excitability. When combined with a loss-of-function mutation in another voltage-gated K⁺ channel, shaker (sh), the eag phenotype is further enhanced, suggesting that both K⁺ channels contribute to the repolarization of presynaptic nerve terminals.^13–16 Recently, Gardnell et al., identified a gain-of-function mutation in the Eag channel that suppresses the effect of sh mutation on behavior and neuronal excitability in Drosophila.¹⁷ The mutation is caused by a single amino acid substitution (G297E) in the S2–S3 linker of the Eag channel protein. Interestingly, they have determined that extracellular Mg²⁺ is required for the gain-of-function eag mutant to suppress sh phenotypes. It thus raises the possibility that the G297E mutation may affect Eag channel properties regulated by the divalent ion.Eag channels are uniquely regulated by a variety of external divalent cations, including Mg²⁺, Mn²⁺, and Ni²⁺.^18–21 The major effect of these divalent ions is to slow activation kinetics, which presumably reflects a switch of Eag channels from a fast to a slow mode of activation gating.²² Based on previous studies, the divalent ions appear to modulate activation process of the Eag K⁺ channel through binding to acidic residues located in an extracellular- facing crevice between transmembrane segments S2 and S3 of the voltage sensor domain.^20,23,24 However, the molecular and physical mechanisms underlying divalent ion-dependent switch of gating modes in the Eag channel, particularly how does ion binding affects voltage sensor and its coupling to the activation gate, are yet to be determined.^24,25In this study, we carried out detailed analysis of the G297E mutant Eag channel in a heterologous expression system. Our data reveal that this mutation resulted in a significant decrease in Mg²⁺-induced deceleration of Eag channel activation kinetics, which provided a mechanistic basis for its gain-of-function phenotype in flies. In addition, we identified another mutation in the other intracellular linker that, in conjunction with G297E, eliminated Mg²⁺-dependent regulation of channel activation. Our findings set a stage for further determination of molecular detail of Eag channel activation processes.     

Private channels in plant-pollinator mutualisms

Volatile compounds often mediate plant-pollinator interactions, and may promote specialization in plant-pollinator relationships, notably through private channels of unusual compounds. Nevertheless, the existence of private channels, i.e., the potential for exclusive communication via unique signals and receptors, is still debated in the literature. Interactions between figs and their pollinating wasps offer opportunities for exploring this concept. Several experiments have demonstrated that chemical mediation is crucial in ensuring the encounter between figs and their species-specific pollinators. Indeed, chemical messages emitted by figs are notably species- and developmental stage-specific, making them reliable cues for the pollinator. In most cases, the species-specificity of wasp attraction is unlikely to result from the presence of a single specific compound. Nevertheless, a recent paper on the role of scents in the interaction between Ficus semicordata and its pollinating wasp Ceratosolen gravelyi showed that a single compound, 4-methylanisole, is the main signal compound in the floral scent, and is sufficient by itself to attract the obligate pollinator. Mainly focusing on these results, we propose here that a floral scent can act as a private channel, attracting only the highly specific pollinator.Key words: chemical mediation, ficus, agaonidae, private channel, floral filter, coevolved mutualismMutualisms are interspecies interactions in which each participant gains net benefits from interacting with its partner. Like many other interspecies interactions, mutualisms are usually mediated by chemical signals. For instance, floral scents act as pollinator attractants in numerous plant species.^1–3 We studied the chemical compounds that mediate a set of interactions which has become a model system for understanding the evolution of mutualisms: the interactions between Ficus (Moraceae) and their species-specific pollinating fig wasps (Chalcidoidea: Agaonidae). In this ‘nursery pollination mutualism’, the pollinators can breed only in receptive figs of their host tree, which depends in turn on the wasp as its sole pollinator. Each pollinator species is usually associated with a single Ficus species. Fig trees mainly grow in tropical regions, and many species can co-occur in the same forest. In these regions, the density of individual species is often quite low.⁴ Therefore, signals emitted by each species must be efficient at long distances and specific, to allow the attraction of the associated pollinator. In all of the Ficus species that have been studied so far (approximately 40 of a total of roughly 800 species worldwide), figs have been shown to release volatile compounds when they are receptive (i.e., at the stage when pollination can occur).^5–10 Behavioral tests have been performed with pollinators of several species, confirming for these species the role of fig scent in pollinator attraction (Soler et al. in prep).^5–7,9,11 In the floral scents of the fig species studied, at least two to five major compounds account for the majority of the total volatiles emitted by receptive figs.^8,10,12–15 These major volatiles emitted by receptive figs are generally compounds that are not rare in floral fragrances. The species-specificity of wasp attraction is thus usually not likely to result from the presence of only one single specific compound.^10,13 However, Chen and co-workers,¹⁶ focusing on Ficus semicordata, found that a single benzenoid compound, 4-methylanisole, is sufficient to attract its pollinator. Though 4-methylanisole occurs in floral fragrances, having been documented in floral scents of plants from 17 families (of the 90 families included in the review by Knudsen et al.),¹⁷ it usually accounts for only a fraction of total volatiles, and this was the first time that this benzenoid compound has been reported in the floral scent of a Ficus species.^7,10–12,18 To our knowledge, no previous study has shown that 4-methylanisole is attractive to pollinators of any plant, or that this compound could by itself mediate the specificity of any mutualistic interaction.Raguso (2008)³ defined a ‘private channel’ as the potential for such exclusive communication via unique signals and receptors. Moreover, according to Schaefer et al.¹⁹ private channels must fit three major criteria: (1) the identification of an intended (effective mutualist) receiver; (2) sensitive signal detection by this receiver, and finally (3) poor detection by unintended (less effective) receivers. Chen et al.¹⁶ showed that a single compound, 4-methylanisole, accounted for more than 90% of the volatile compounds emitted by receptive figs of F. semicordata. This compound is also known not to be produced by the two other sympatric Ficus species in which floral odours have also been studied, nor by any Ficus species whose scent has been analyzed (Soler et al., in preparation).^10,13 Moreover, Chen et al.¹⁶ found that the species-specificity of the attraction of Ceratosolen gravelyi, the pollinating wasp of Ficus semicordata, was due mainly, if not entirely, to this single major compound. Indeed, based on behavioural (olfactory) tests, they showed that the specialized pollinating wasp detects 4-methylanisole and is attracted by it, even at low concentrations (wasp response tested in concentrations ranging from 1.22 × 10² ng/100 µl to 1.22 × 10⁶ ng/100 µl). The last criterion for a private channel proposed by Schaefer et al.¹⁹ i.e., poor detection by unintended receivers, is the only one which has not been clearly demonstrated by Chen et al.¹⁶ In the case of fig/fig wasp mutualisms, the unintended receivers correspond obviously to the parasites of the mutualism. Indeed, many fig species harbor numerous species of chalcidoid wasps that mature within ovaries in fig inflorescences, like the pollinator, but do not carry pollen. Each of these non-pollinating fig wasps is assumed to be associated specifically with a single Ficus species²⁰ and to use fig scents as cues to detect the host species, as do pollinating fig wasps.²¹ However, in the case of the F. semicordata/C. gravelyi mutualism, no non-pollinating fig wasps have been observed ovipositing during the period when figs are at the receptive stage. This situation is quite unusual in Ficus species (Proffit M, unpublished data; Rasplus J-Y, personal communication). The apparent absence of ‘eavesdropping’ parasites at the time when pollinators are attracted to the figs suggests that the last criterion of a private channel—poor detection by potential receivers other than the specific pollinator—also holds in this case, although this has not been experimentally demonstrated.The study by Chen et al.¹⁶ is, to our knowledge, the first that attempted to test the existence of a private channel in a fig/fig wasp interaction. Nevertheless, this is not the first case of a putative private channel in a nursery pollination mutualism. Indeed, examples have been highlighted in the interactions between Yucca filamentosa (Agavaceae) and its moth pollinator Tegeticula cassandra (Prodoxidae)²² and in the interactions between Peltandra virginica (Araceae) and its pollinating fly Elachiptera formosa (Chloropidae).²³ Similarly, the existence of private channels has been suggested for several species in the family Eupomatiaceae, pollinated by beetles.²⁴ In contrast, Svensson et al.²⁵ showed that in the Breynia (Phyllanthaceae)/Epicephala (Gracillariidae) interaction, pollinators are attracted by common volatile compounds, supporting the hypothesis that no private channel exists in this case. In a review of the role of scents in plant-pollinator interactions, Raguso³ highlighted the putative examples of private channels, but noted that their existence is still debated, notably in the case of non co-specialized plant-pollinator interactions. Nevertheless, some studies have suggested that private channels do exist in this class of interactions. Most examples concern the Orchidaceae. For instance, Eltz et al.²⁶ showed that carvone oxide and ipsdienol are volatile floral rewards emitted by Neotropical orchids pollinated by male euglossine bees, which collect volatile substances for courtship displays. However, probably the best-known examples are Ophrys spp., temperate-zone terrestrial orchids whose flowers mimic the female pheromone of the pollinator to attract males, which pollinate flowers by pseudo-copulation.^27–29 In his review of the role of scents in plant pollinator interactions, Raguso³ also proposed that floral filters do not need to rely upon exclusive olfactory signals or receptors. Indeed, few studies have determined whether pollinator-attractive compounds could alone assure species-specificity (private channel), or whether specificity is mediated by more complex ‘floral filters’, of which scent is only one component. These latter may integrate mechanical or other kinds of barriers, as seems to be the case in the interaction between the dwarf palm Chamaerops humilis and the weevil Derelomus chamaeropsis.³⁰In the literature about private channels in chemical mediation of mutualisms, two points still seem unclear. One is a semantic point: do cases in which specificity is ensured by specific ratios of several more common compounds constitute a private channel, or is this concept restricted to cases in which specificity is ensured by a single rare compound? The literature on private channels emphasizes cases of the latter type. A related question is whether specificity is sometimes ensured not by a single rare compound, but by a combination of rare compounds. While all the putative cases of private channels that have attracted attention concern emission of and attraction to a single rare compound,^3,16,18 the limited number of studied cases does not allow drawing firm general conclusions. Nevertheless, we might think that a hypothetical private channel constituted by several rare compounds might be more difficult to evolve (if it requires the acquisition of several biosynthetic pathways by the plant, and of specific receptors by the insect) or counter-selected (owing to greater costs).We propose here that in the mutualism between F. semicordata and C. gravelyi, despite the existence of mechanical barriers to flower visitation (as in all Ficus species), available information on the chemical communication between plant and pollinator constitutes a strong case for the mediation of a highly specific interaction through a private channel, which acts largely alone as a floral filter that prevents ‘cheaters’ from finding and exploiting a potential resource. An interesting long-term consequence of such an adaptation in a highly co-specialized plant-pollinator interaction is that it might reduce evolutionary flexibility, preventing host shifts, and perhaps making it difficult for the mutualists to evolve counter-adaptations to a new parasite that ‘decodes’ the private channel. Private channels may be isolated adaptive peaks, even more difficult to escape than they are to reach. This could explain their apparent infrequent occurrence in nature.     

Subcellular localization of overexpressed maize AChE gene in rice plant

The ACh-mediated system consisting of acetylcholine (ACh), acetylcholine receptor (AChR) and acetylcholinesterase (AChE) is fundamental for nervous system function in animals and insects. Although plants lack a nervous system, both ACh and ACh-hydrolyzing activity have been widely recognized in the plant kingdom. The function of the plant ACh-mediated system is still unclear, despite more than 30 years of research. To understand ACh-mediated systems in plants, we previously purified maize AChE and cloned the corresponding gene from maize seedlings (Plant Physiology). In a recent paper in Planta, we also purified and cloned AChE from the legume plant siratro (Macroptilium atropurpureum). In comparison with electric eel AChE, both plant AChEs showed enzymatic properties of both animal AChE and animal butyrylcholinesterase. On the other hand, based on Pfam protein family analysis, both plant AChEs contain a consensus sequence of the lipase GDSL family, while the animal AChEs possess a distinct alpha/beta-hydrolase fold superfamily sequence, but no lipase GDSL sequence. Thus, neither plant AChE belongs to the well-known AChE family, which is distributed throughout the animal kingdom. To address the possible physiological roles of plant AChEs, we herein report our data from the immunological analysis of the overexpressed maize AChE gene in plants.Key words: acetylcholinesterase activity, maize AChE gene, overexpression, rice, subcellular localizationIn the animal ACh-mediated system, ACh serves to propagate an electrical stimulus across the synaptic junction. At the presynaptic neuron end, an electrical impulse triggers the release of ACh, which accumulates in vesicles into the synaptic cleft via exocytosis. ACh then binds to an ACh receptor (AChR) on the postsynaptic neuron surface, and the ACh-AChR binding induces subsequent impulses to the postsynaptic neuron. Finally, ACh, which is released again by the receptor into the synaptic cleft, is rapidly degraded by acetylcholinesterase (AChE; E.C.3.1.1.7).^1,2 ACh and AChE,^3,4,5 and choline acetyltransferase activity that takes part during synthesis of ACh^6,7 have been recognized in plants. AChR has not been identified in plant cells so far. However, so-called “ACh-binding sites” were detected in membrane fractions from some bean seedlings^8,9 and evidence was also detected in plant organelles, such as chloroplasts¹⁰ and tonoplasts¹¹ using pharmacological methods.Concerning the function of the ACh-mediated system in plants, Momonoki^12,13 has proposed that it results in an asymmetric distribution of hormones and substances due to gravity stimuli, as well as changes in ACh content, AChE activity and Ca²⁺ level in response to heat stress. However, all these phenomena have been investigated using indirect techniques. Thus, to understand the plant ACh-mediated system, we purified AChEs and cloned the AChE genes from maize¹⁴ and siratro¹⁵ seedlings. The maize AChE was found to exist as two types of 88-kDa homodimers, which in turn consisted of disulfide-linked and noncovalently-linked 42- to 44-kDa subunits.¹⁴ The siratro AChE might exist as a disulfide-linked 125-kDa homotrimer consisting of 41- or 42-kDa subunits.¹⁵ The plant AChEs apparently from various quaternary structures, depending on the plant species, similar to animal AChEs. Furthermore, maize and siratro AChEs showed enzymatic properties of both animal AChE and animal butyrylcholinesterase, compared with electric eel AChE.¹⁵In this addendum, we report our recent immunohistochemical study using an antibody against maize AChE. In order to overexpress the maize AChE gene in rice plants, we constructed a plasmid for the sense expression of the AChE gene by cloning it into the pT7 Blue vector. The maize AChE gene¹⁴ was introduced behind the maize ubiquitin 1 promoter (Ubi) in the p2K-1⁺ plant expression vector. The Ubi::maize AChE and control (p2K-1⁺ only) plasmid were introduced into Agrobacterium tumefaciens EHA 101, which was transformed into rice (Nihonbare) via Agrobacterium-mediated transformation methods.¹⁶ The maize AChE transgenic plants exhibited approximately 650-fold higher AChE activity than was observed in the control plants but no phenotypic changes between transgenic and control plants. The subcellular localization of AChE was observed by immunofluorescence in paraffin-embedded leaf and stem tissues of transgenic rice plants. The maize AChE protein was detected in extracellular spaces in the leaf and stem of the plants (Fig. 1). Therefore, plant AChEs may function in the extracellular space, similar to some isoforms of animal AChE.^2,17Open in a separate windowFigure 1Subcellular localization of maize AChE in leaf and stem of transgenic rice. (A) Leaf cross-section of transgenic rice; (B) leaf cross-section of control; (C) stem cross-section of transgenic rice; (D) stem cross-section of control. Each section was probed with maize AChE antibody and then visualized with Alexa Fluor 488-conjugated secondary antibody. Control indicates rice plants transfected with p2K-1⁺ vector only. Arrowheads indicate localization of maize AChE.Most of the AChE activity in the root was associated with cell wall materials.¹⁸ The computer-assisted cellular sorting prediction program TargetP presumed that our purified maize AChE¹⁴ is targeted to the secretory pathway via the endoplasmic reticulum. Furthermore, the SOSUI program (http://sosui.proteome.bio.tuat.ac.jp / sosuiframe0.html), which discriminates between membrane and soluble proteins, showed that the maize AChE does not contain any likely transmembrane helical regions, which are features of proteins that associate with the lipid bilayers of the cell membrane. These findings suggested that the maize AChE might be localized at the cell wall. However, in an earlier work,¹³ we speculated that AChE is localized at the plasmodesmatal cell-to-cell interface and that it plays a role in regulation of the plasmodesmatal channel as a constituent of the ACh-mediated system. We improved our hypothesis of the role of the ACh-mediated system in a paper in Plant Physiol.¹⁴The results based on fluorescence-immunohistochemistry in transgenic rice plants reported in this paper confirmed that the maize AChE is localized at the cell wall. Here we propose again our hypothesis of an ACh-mediated system including this new finding; the system might be localized to the extracellular region around the plasmodesmatal channel and might conduct cell-to-cell trafficking by channel gating regulation. Adjoining cells in plant tissues are interconnected via plasmodesmata, which allow the trafficking of low-molecular-mass materials across the cell wall between two cells. According to a recent model,¹⁹ transport of these substances could be regulated by the opening and/or closing of conductive channels to prevent infection by pathogens and to selectively control trafficking through the plasmodesmata. Furthermore, it has been speculated that morphoregulatory proteins around the plasmodesmata could be involved in channel regulation.²⁰ Therefore, the ACh-mediated system might regulate the opening and/or closing of channels by interactions with morphoregulatory proteins at the cell wall matrix surrounding the plasmodesmata. Further research will be required to clarify the precise physiological roles of plant AChEs.     

Filamentous brown algae infected by the marine,holocarpic oomycete Eurychasma dicksonii: first results on the organization and the role of cytoskeleton in both host and parasite

The important role of the cytoskeletal scaffold is increasingly recognized in host-pathogen interactions. The cytoskeleton potentially functions as a weapon for both the plants defending themselves against fungal or oomycete parasites, and for the pathogens trying to overcome the resisting barrier of the plants. This concept, however, had not been investigated in marine algae so far. We are opening this scientific chapter with our study on the functional implications of the cytoskeleton in 3 filamentous brown algal species infected by the marine oomycete Eurychasma dicksonii. Our observations suggest that the cytoskeleton is involved in host defense responses and in fundamental developmental stages of E. dicksonii in its algal host.Oomycetes are important plant and animal pathogens and are the cause of significant crop losses every year. Hence, a plethora of studies with different cultivated and model plant species investigate the diversity of parasite infection pathways and host defense responses.¹ However, little information is available on the interactions between algae and marine oomycetes, despite the epidemic outbreaks reported² and the huge impact on intensive algal aquaculture.³Eurychasma dicksonii is a biotrophic, intracellular marine oomycete, capable to infect at least 45 species of brown seaweeds in laboratory cultures.⁴ Molecular data reveal that E. dicksonii has a basal phylogenetic position in the oomycete lineage.^5,6 The basic stages of the infection are known: the attachment of the parasite spore to the host cell wall, the penetration of its cytoplasm into the host cell, the formation of a multinucleated, unwalled thallus, and zoosporogenesis.⁶ Hitherto, though, there was no knowledge about the role of cytoskeleton in the context of infection, which stimulated our research.In land plants, reorganization of the cytoskeleton is part of the reaction to infection by fungal pathogens. The rearrangement of the cytoplasm and the relocation of the nuclei and other organelles are accompanied by rapid rearrangements of the cytoskeletal elements.⁷ The plant cytoskeleton shows an extreme plasticity in order to serve the intracellular realignment.At the same time, this indicates that the plant cytoskeleton could be the parasite’s target by producing anti-cytoskeletal compounds in an effort to overcome plant resistance, a mechanism known in several fungal and oomycete pathogens of higher plants.^8,9Consequently, the changes in microtubule (MT) organization are associated with both the plant defense and/or susceptibility toward oomycetes, respectively.¹⁰ Therefore, our research on the organization and role of cytoskeleton in the host and the parasite sheds some light into the enormous variability in the specificity of the recognition, defense, and infection mechanisms.     

AtMSL9 and AtMSL10: Sensors of plasma membrane tension in Arabidopsis roots

Plant cells, like those of animals and bacteria, are able to sense physical deformation of the plasma membrane. Mechanosensitive (MS) channels are proteins that transduce mechanical force into ion flux, providing a mechanism for the perception of mechanical stimuli such as sound, touch and osmotic pressure. We recently identified AtMSL9 and AtMSL10, two mechanosensitive channels in Arabidopsis thaliana, as molecular candidates for mechanosensing in higher plants.¹ AtMSL9 and AtMSL10 are members of a family of proteins in Arabidopsis that are related to the bacterial MS channel MscS, termed MscS-Like (or MSL).² MscS (Mechanosensitive channel of Small conductance) is one of the best-characterized MS channels, first identified as an electrophysiological activity in the plasma membrane (PM) of giant E. coli spheroplasts.^3,4 Activation of MscS is voltage-independent, but responds directly to tension applied to the membrane and does not require other cellular proteins for this regulation.^5,6 MscS family members are widely distributed throughout bacterial and archaeal genomes, are present in all plant genomes yet examined, and are found in selected fungal genomes.^2,7,8 MscS homolgues have not yet been identified in animals.Key words: Arabidopsis thaliana, root, MscS, MSL, plasma membrane, mechanotransductionWe previously showed that in wild type protoplasts from the Arabidopsis root, AtMSL9 and AtMSL10 function cooperatively to provide a characteristic WT activity. In this paper, we further investigate the function of AtMSL9 and AtMSL10. We analyze individual protoplasts and argue that in WT cells AtMSL9 and AtMSL10 can function either in cooperation or independently. We also compare the electrophysiological properties of these two channels with that of their bacterial and algal counterparts, and discuss their possible function in planta.     

Piriformospora indica mycorrhization increases grain yield by accelerating early development of barley plants

Root colonization by the basidiomycete fungus Piriformospora indica induces host plant tolerance against abiotic and biotic stress, and enhances growth and yield. As P. indica has a broad host range, it has been established as a model system to study beneficial plant-microbe interactions. Moreover, its properties led to the assumption that P. indica shows potential for application in crop plant production. Therefore, possible mechanisms of P. indica improving host plant yield were tested in outdoor experiments: Induction of higher grain yield in barley was independent of elevated pathogen levels and independent of different phosphate fertilization levels. In contrast to the arbuscular mycorrhiza fungus Glomus mosseae total phosphate contents of host plant roots and shoots were not significantly affected by P. indica. Analysis of plant development and yield parameters indicated that positive effects of P. indica on grain yield are due to accelerated growth of barley plants early in development.Key words: mycorrhiza, barley development, Piriformospora indica, phosphate uptake, grain yield, pathogen resistanceThe wide majority of plant roots in natural ecosystems is associated with fungi, which very often play an important role for the host plants'' fitness.¹ The widespread arbuscular mycorrhizal (AM) symbiosis formed by fungi of the phylum Glomeromycota is mainly characterized by providing phosphate to their host plant in exchange for carbohydrates.^2,3 Fungi of the order Sebacinales also form beneficial interactions with plant roots and Piriformospora indica is the best-studied example of this group.⁴ This endophyte was originally identified in the rhizosphere of shrubs in the Indian Thar desert,⁵ but it turned out that the fungus colonizes roots of a very broad range of mono- and dicotyledonous plants,⁶ including major crop plants.^7–9 Like other mutualistic endophytes, P. indica colonizes roots in an asymptomatic manner¹⁰ and promotes growth in several tested plant species.^6,11,12 The root endophyte, moreover, enhances yield in barley and tomato and increases in both plants resistance against biotic stresses,^7,9 suggesting that application in agri- and horticulture could be successful.     

Potassium flux in the pollen tubes was essential in plant sexual reproduction

Potassium channels are controlling K⁺ transport across plasma membrane and thus playing a central role in all aspects of osmolarity as well as numerous other functions in plants, including in sexual reproduction. We have used whole-cell and single-channel patch-clamp recording techniques investigated the regulation of intracellular free Ca²⁺-activated outward K⁺ channels in Pyrus pyrifolia pollen tube protoplasts. We have also showed the channels could be inhibited by heme and activated carbon monoxide (CO). In the presence of oxygen and NADPH, hemoxygenases catalyzes heme degradation, producing biliverdin, iron and CO. Considered the oxygen concentration approaching zero in the ovary, the heme will inhibit the K⁺ outward flux from the intracellular of pollen tube, increasing the pollen tubes osmolarity, inducing pollen tube burst. Here we discuss the putative role of K⁺ channels in plant sexual reproduction.Key words: pear, pollen, K⁺ channels, heme, carbon monoxideIon channels in the pollen tube play critical roles in mediation pollen germination and pollen tube growth.^1–3 Early studies were focus on the plasma membrane calcium channel regulation and cytosolic free calcium concentration variation in the pollen tube reason by which was one of the most important second messengers in plants.^3–7 However, reports have also showed that the potassium channels in the pollen tubes were also involved in several important steps of plant sexual reproduction.^8–19 Recently, more reports further demonstrated this phenomena.^20–24 In the report by Lu et al. they demonstrated that two cation/proton exchangers (CHX), CHX21 and CHX23, are essential for pollen tube growth guidance in Arabidopsis.²² chx21 chx23 double mutant induces the fertility impaired, but which is unchanged in both single chx21 or chx23 mutants. They have also found that the double mutant pollen grains germination and pollen tube growth in the transmitting tract were not difference with the wild-type, however, the double mutant pollen tubes fail to turn toward ovules.²² Protein localization experiments show CHX23 is expressed in the endoplasmic reticulum of pollen tubes; functional analysis results showed that CHX23 as a K⁺ transporter mediates K⁺ uptake in a pH-dependent manner. So, these protein affect the signal transduction pathway of pollen tube growth toward to the ovule by controlling the cation balance and pH in the pollen tube.²² Amien et al. identified a signaling ligand of defensin-like (DEFL) protein, ZmES4, which expressed in maize synergid. ZmES4 activates the maize pollen tube tip plasma membrane K⁺ Shaker channel KZM1.²⁰ This finding is also very interesting. Pollen tube bursting suggested to be based on the osmotic stress; the influx of K⁺ mediated by ZmES4-activated KZM1 will trigger rapid plasma membrane depolarization, which induced the pollen tube tip burst.²⁰ Furthermore, the osmotic increasing induced by too much K⁺ in the cytosolic of pollen tube was not only resulted by inward K⁺ channel activation, but also resulted by outward K⁺ channel inhibition in the pollen tube plasma membrane. In our report, we find a intracellular Ca²⁺-sensitive outward K⁺ channel in pear pollen tube plasma membrane, which could be inhibited by heme and activated by heme oxidative production, carbon monoxide (CO), may play a functional role in the pollen tube brusting.²³In the presence of oxygen and NADPH, hemoxygenases catalyzes heme degradation, producing biliverdin, iron and CO.²⁵ Early reports showed that oxygen plays an important role in plant sexual reproduction. Pollen tubes grow through the style toward the ovary with high speed, a process that consumes tremendous amounts of energy and requires rapid oxygen uptake by pollen tubes.²⁶ Pollen grains have roughly 20 times the level of mitochondria and respire 10 times faster than vegetative tissue.^12,27–29 Furthermore, oxygen has been proposed as a possible cue for pollen-tube guidance.³⁰ Indeed, the existence of an oxygen gradient in the unpollinated style has been shown in some species such as Hipeastrum hybridum. Oxygen pressure is high in the stigma and style but suddenly decreases at the base of the style, approaching zero in the ovary. Moreover, pollen-tube growth itself creates hypoxic regions within the style.³¹ Therefore, we suggest that the outward K⁺ channel inhibited by heme is dominant compared with which activated by CO when pollen tubes reach the ovary, based on where the hypoxic condition (Fig. 1). However, the gene encode the outward K⁺ channel in the pear pollen tube remains to be determined in the further study.Open in a separate windowFigure 1Reciprocal regulation of heme and carbon monoxide in putative Ca²⁺-activated outward K⁺ channel. Under normal condition, in the presence of NADPH, heme is metabolized by hemeoxygenase to generate carbon monoxide (CO), which activates outward K⁺ channel. However, without the oxygen, heme cannot be metabolized. The accumulated heme acts as an inhibitor of outward K⁺ channel, even in the presence of NADPH. The accumulated K⁺ in the cytosolic of pollen will induced the pollen tube depolarized, then burst.     

Copper-induced calcium release from ER involves the activation of ryanodine-sensitive and IP3-sensitive channels in Ulva compressa

The marine alga Ulva compressa (Chlorophyta) showed a triphasic release of intracellular calcium with maximal levels at 2, 3 and 12 h and a biphasic accumulation of intracellular hydrogen peroxide with peaks at 3 and 12 h when cultivated with copper excess. Intracellular hydrogen peroxide originated exclusively in organelles. In this work, we analyzed the intracellular origin of calcium release and the type of calcium channels activated in response to copper excess. U. compressa was treated with thapsigargin, an inhibitor of endoplasmic reticulum (ER) calcium ATPase, ryanodine, an inhibitor of ryanodine-sensitive channels and xestospongin C, an inhibitor of inositol 1, 4, 5-triphosphate (IP₃)-sensitive channels. Thapsigargin induced the depletion of calcium stored in ER at 75 min and completely inhibited calcium release at 2, 3 and 12 h of copper exposure indicating that calcium release originated in ER. In addition, ryanodine and xestospogin C inhibited calcium release at 2 and 3 h of copper exposure whereas the peak at 12 h was only inhibited by ryanodine. Thus, copper induced the activation of ryanodine-sensitive and IP₃-sensitive calcium channels in ER of U. compressa.Key words: calcium release, endoplasmic reticulum, calcium channels, marine alga, Ulva compressaPlants showed common responses to biotic and abiotic stresses, mainly the accumulation of reactive oxygen species (ROS), in particular hydrogen peroxide, and the release of intracellular calcium.^1,2 Regarding abiotic stress, it has been shown that ozone triggers a NADPH oxidase-dependent biphasic oxidative burst in Arabidopsis thaliana that activates antioxidant and defense enzymes.^3,4 In addition, cadmium induced a NADPH oxidase-dependent monophasic accumulation of extracellular hydrogen peroxide in tobacco cells.⁵ On the other hand, ozone as well as absicic acid treatment, dessication, cold, heat, salinity, UV light and anoxia induce intracellular calcium release and the activation of antioxidant enzymes.^6–8 Regarding abiotic stress in algae, copper induced a monophasic increase of intracellular hydrogen peroxide at 2 h of copper exposure in the brown seaweeds Lessonia nigrecsens and Scytosiphon lomentaria.⁹ On the other hand, strontium induced calcium release in the green microalga Eremosphaera viridis as did osmotic stress in the zygote of the brown macroalga Fucus serratus.^10,11 U. compressa is a cosmopolitan marine macroalga (Chlorophyta) growing in copper-impacted coastal areas in northern Chile.¹² U. compressa cultivated in seawater with copper excess (10 µM) showed co-occuring increases of intracellular calcium and hydrogen peroxide.¹³ Copper induced a triphasic release of calcium with maximal levels at 2, 3 and 12 h and a biphasic production of hydrogen peroxide with peaks at 3 and 12 h. Interestingly, the production of hydrogen peroxide occurred exclusively in organelles, i.e., mitochondria and chloroplasts. In addition, calcium and hydrogen peroxide act as signals in the differential activation of antioxidant and defense enzymes.¹³ In this work, we analyzed the intracellular origin of copper-induced calcium release and the type of calcium channels activated in response to copper excess in U. compressa.     

Müllerian mimicry in aposematic spiny plants

Müllerian mimicry is common in aposematic animals but till recently, like other aspects of plant aposematism was almost unknown. Many thorny, spiny and prickly plants are considered aposematic because their sharp defensive structures are colorful and conspicuous. Many of these spiny plant species (e.g., cacti and Agave in North American deserts; Aloe, Euphorbia and acacias with white thorns in Africa; spiny plants in Ohio; and spiny members of the Asteraceae in the Mediterranean basin) have overlapping territories, and also similar patterns of conspicuous coloration, and suffer from the evolutionary pressure of grazing by the same large herbivores. I propose that many of these species form Müllerian mimicry rings.Key words: aposematic coloration, defense, evolution, herbivory, müllerian mimicry, spines, thornsAposematic (warning) coloration is a biological phenomenon in which poisonous, dangerous or otherwise unpalatable organisms visually advertise these qualities to other animals. The evolution of aposematic coloration is based on the ability of target enemies to associate the visual signal with the risk, damage or non-profitable handling, and later to avoid such organisms as prey. Typical colors of aposematic animals are yellow, orange, red, purple, black, white or brown and combinations of these.^1–5 Many thorny, spiny and prickly plant species were proposed to be aposematic because their sharp defensive structures are usually colorful (yellow, orange, red, brown, black, white) and/or associated with similar conspicuous coloration.^5–22 Animal spines also have similar conspicuous coloration and were proposed to be aposematic.^1,5,17,23Several authors have proposed that mimicry of various types helps in plant defense, e.g.,^9,24–34 More specifically, Müllerian mimicry was already proposed to exist in several defensive plant signaling systems. The first was for several spiny species with white-variegated leaves.^8,10 The second was for some tree species with red or yellow poisonous autumn leaves.³⁵ The third cases are of a mixture of Müllerian and Batesian mimicry, of thorn auto-mimicry found in many Agave species.⁸Here I propose that many species of visually aposematic spiny plants of the following taxa: (1) Cactaceae, (2) the genus Agave, (3) the genus Aloe, (4) African thorny members of the genus Euphorbia, (5) African acacias with white thorns, (6) spiny vascular plants of southeastern Ohio, (7) spiny Near Eastern plants with white variegation on their leaves, (8) Near Eastern members of the Asteraceae with yellow spines, form Müllerian mimicry rings of spiny plants.To consider the existence of Müllerian mimicry rings in aposematic organisms, two factors are needed: (1) a similar signal, and (2) an overlapping distribution in respect to the territory of predators in animals, or herbivores in plants. I will show below that for the plant taxa proposed here to form Müllerian mimicry rings, both criteria operate.The accumulating data about the common association of plant defenses by spines with visual conspicuousness, along with the fact that many such species overlap in their habitat, raises the possibility of the broad phenomenon of existence of Müllerian mimicry rings in plants. Even from the limited number of publications proposing visual aposematism in spiny plants, the operation of vegetal Müllerian mimicry rings seems to be obvious. The phenomenon can now be traced to both the Old World (Asia, Africa and Europe) and the New World (North America). The best-studied cases include Cactaceae and the genera Agave, Aloe and Euphorbia,⁶ African acacias with white thorns,^12,15 Near Eastern spiny plants with white variegation on their leaves,^7,11 aposematic spiny vascular plants of southeastern Ohio,¹⁶ and many spiny Mediterranean species of the Asteraceae with yellow spines.²²In the four spiny taxa (Cactaceae and the genera Agave, Aloe and Euphorbia) that were the first to be proposed as visually aposematic⁶ there is a very strong morphological similarity. In cacti, there are two types of conspicuousness of spines that are typical of many plant species: (1) colorful spines, and (2) white spots, or white or colorful stripes, associated with spines on the stems. These two types of aposematic coloration also dominate the spine system of Agave, Aloe and Euphorbia. The fact that many species of three of these four spiny taxa (Agave, Aloe and Euphorbia) are also poisonous^36–38 further indicates their potential to form Müllerian mimicry rings.I propose that each of these groups for itself and some of these groups (e.g., Cactaceae and the genus Agave in North America; Aloe, Euphorbia and acacias in east and south Africa) that have overlapping distribution and share at least some of the herbivores, form Müllerian mimicry rings.The first Müllerian mimicry ring is of cacti and Agave that have an overlapping distribution over large areas in North America.^37,39 The large herbivores in North America disappeared not so long ago in evolutionary time scales and seem to have shaped the spiny defense of these plant taxa.⁴⁰The second Müllerian mimicry ring is of the spiny and thorny members of the African genera Aloe, Euphorbia and certain acacias with very conspicuous white thorns, which partly overlap in distribution and share various large mammalian herbivores.^12,15,36,41The third Müllerian mimicry ring is the outcome of the common presence of aposematic coloration in spiny vascular plants of southeastern Ohio,¹⁶ with color patterns in thorns and spines similar to those of Cactaceae and the genera Agave, Aloe and Euphorbia described in Lev-Yadun.⁶The next case of potential operation of Müllerian mimicry ring of spiny plants with overlapping territories that suffer from the same large herbivores, but on a much smaller geographical scale, has recently been proposed for several spiny species with white-variegated leaves,⁷ and later for more than 20 spiny species in the flora of Israel that have white markings associated with their spines.¹¹The last case of a probable Müllerian mimicry ring was described by Ronel et al.²² who while studying the spine system of Near Eastern spiny members of the Asteraceae, found 29 spiny species with yellow spines, and additional such species are expected to occur. Since some of these species and others with yellow spines also grow in southern Europe, it is clear that the same phenomenon is also common there.I conclude that Müllerian mimicry rings seem to be very common in plants, and that it is probable that many other spiny plants that form Müllerian mimicry rings are waiting to be studied. Such defensive rings are probably also formed by poisonous plants that share similar colors or odors.     

Creating drought- and salt-tolerant cotton by overexpressing a vacuolar pyrophosphatase gene

Increased expression of an Arabidopsis vacuolar pyrophosphatase gene, AVP1, leads to increased drought and salt tolerance in transgenic plants, which has been demonstrated in laboratory and field conditions. The molecular mechanism of AVP1-mediated drought resistance is likely due to increased proton pump activity of the vacuolar pyrophosphatase, which generates a higher proton electrochemical gradient across the vacuolar membrane, leading to lower water potential in the plant vacuole and higher secondary transporter activities that prevent ion accumulation to toxic levels in the cytoplasm. Additionally, overexpression of AVP1 appears to stimulate auxin polar transport, which in turn stimulates root development. The larger root system allows AVP1-overexpressing plants to absorb water more efficiently under drought and saline conditions, resulting in stress tolerance and increased yields. Multi-year field-trial data indicate that overexpression of AVP1 in cotton leads to at least 20% more fiber yield than wild-type control plants in dry-land conditions, which highlights the potential use of AVP1 in improving drought tolerance in crops in arid and semiarid areas of the world.Key words: drought tolerance, proton pump, salt tolerance, transgenic cotton, vacuolar membraneDrought and salinity are major environmental factors that limit agricultural productivity in most parts of the world.¹ Climate change will likely make many places worse in terms of water availability and soil salinization,² which will have negative impacts on food production in world agriculture. Yet, the demand for more food will continue to rise because of the growing world population that may reach 9 billon people by 2050.³ Therefore, the primary challenge we face during this century is the production of more food under the constraints of limited water and fertilizer on marginal soils.Many genes that respond to abiotic stresses have been identified in the model plant Arabidopsis,⁴ and some of them were shown to play important roles in protecting plants under abiotic stress conditions.⁵ The Arabidopsis vacuolar pyrophosphatase gene AVP1 appears to be one of the most promising genes that may be used to improve drought- and salt-tolerance in crops.⁶ Roberto Gaxiola''s group first demonstrated that overexpression of AVP1 could lead to significantly improved drought- and salt-tolerance in transgenic Arabidopsis plants.⁷ Later when this gene was introduced into tomato⁸ and rice,⁹ similar tolerance phenotypes were observed. Overexpression of AVP1 in cotton, not only improved drought- and salt-tolerance in greenhouse conditions, but also increased fiber yield in dryland field conditions.⁶ AVP1-expressing cotton plants produced larger root systems and bigger shoot biomass than controls when grown under hydroponic conditions in the presence of up to 200 mM NaCl.⁶ In the greenhouse, AVP1-expressing cotton plants also produced more root and shoot biomass than controls when grown under saline conditions or reduced irrigation.⁶ The increased yield by AVP1-expressing cotton plants is due to more bolls produced, which in turn is due to larger shoot system that AVP1-expressing cotton plants develop under saline or drought conditions.⁶The larger root systems of AVP1-expressing cotton plants under saline and water-deficit conditions allow transgenic plants access to more of the soil profile and available soil water resulting in increased biomass production and yield. Li et al. showed that the larger root systems of AVP1-overexpressing Arabidopsis is caused by increased auxin polar transport in the root, which stimulates root development in AVP1-overexpressing Arabidopsis plants.¹⁰ Furthermore, a recent comparative study of transgenic Arabidopsis lines that produce enlarged leaves showed that auxin levels were increased by 50% in AVP1-overexpressing plants.¹¹ To test if altered auxin level is responsible for the observed larger root systems in AVP1-expressing cotton plants, we germinated wild-type and AVP1-expressing cotton plants in the absence or presence of the auxin polar transport inhibitor Naphthylphthalamic acid (NPA). Both wild-type and AVP1-expressing cotton plants developed robust lateral root systems in the absence of NPA (Fig. 1A). The presence of 50 µM NPA resulted in nearly complete inhibition of lateral root development in wild-type plants, while lateral root development in AVP1-expressing plants was reduced, it was significantly greater than wild-type (Fig. 1B). These data indicate that AVP1-overexpression could overcome the inhibitory effects of NPA on root development in AVP1-expressing cotton plants, suggesting that either increased auxin transport or higher auxin concentration in the root systems of AVP1-expressing cotton plants is responsible for the observed larger root systems, and eventually for the increased boll numbers and fiber yields under dryland field conditions.Open in a separate windowFigure 1Root development of wild-type and AVP1-expressing cotton plants in the absence and presence of auxin transport inhibitor NPA. (A) Phenotype of cotton roots after 10 days of growth in the absence of NPA. WT, Wild-type; 1, 5, 9, three independent AVP1-overexpressing cotton lines. (B) Phenotype of cotton roots after 10 days of growth in the presence of 50 µm NPA.Many genes that may play important roles under water-deficit conditions have been tested in laboratory conditions,^4,5 but very few have been tested vigorously in field conditions. A bacterial cold shock protein gene was shown to improve drought tolerance in maize based on multi-year and multi-place field trial experiments,¹² and it appears that this gene will likely gain approval for commercial release and become the first genetically engineered product that demonstrates improved drought tolerance in a major crop in the U.S. Another example of increased drought tolerance supported by multiple field trial experiments is through downregulation of farnesylation in transgenic canola plants.¹³ Downregulation of farnesyltransferase by antisense or RNAi techniques in transgenic canola leads to increased sensitivity to abscisic acid, consequently resulting in smaller guard cell aperture under drought conditions. These transgenic canola plants lose less water through transpiration and are more drought resistant. Data from more than 5 years of field studies in Canada consistently proved that this approach can indeed increase drought tolerance in transgenic canola. Our study with AVP1-expressing cotton over the last several years in field conditions is another example that genetic engineering approach can be an efficient tool in generating drought-tolerant crops. AVP1-expressing cotton plants can establish a larger shoot mass in dryland conditions (Fig. 2), which results in increased boll numbers and fiber production. Our approach is likely applicable to other major crops as well.Open in a separate windowFigure 2Wild-type and AVP1-expressing cotton plants grown in the dryland field condition. Plants were planted in the middle of may 2009 and the picture was taken in the middle of July 2009 at the USDA experimental Farm in Lubbock, Texas.     

The role of vacuolar processing enzymes in plant immunity

Proteases play important roles in plant innate immunity. In this mini-review, we describe the current view on the role of a plant protease, vacuolar processing enzyme (VPE), and the first identified plant caspase-1-like protein, in plant immunity. In the past several years, VPEs were determined to play important roles in various types of cell death in plants. Early studies demonstrated the identification of VPE as a vacuolar hydrolytic protein responsible for maturation of vacuolar proteins. Later, Nicotiana benthamiana VPE was reported to mediate virus-induced hypersensitive response by regulating membrane collapse. The ortholog of VPE in Arabidopsis is also suggested to be involved in both mycotoxin-induced cell death and developmental cell death. However, the role of VPE in elicitor-signaling is still unclear. Our recent studies demonstrated the involvement of VPE in elicitor signal transduction to induce stomatal closure and defense responses, including defense gene expression and hypersensitive cell death.Key words: hypersensitive cell death, elicitor, stomatal closure, pathogen-associated molecular patterns, plant innate immunity, programmed cell deathIn the course of their development, plants have had to face a wide range of potential pathogens, including viral, bacterial, fungal and oomycete pathogens. Plants, unlike animals, which have specialized defender cells and an adaptive immune system, have an innate immunity of each cell and produce systemic signals emanating from the infection site. The plant innate immunity (PTI) is induced by pathogen-associated molecular patterns (PAMPs)¹ and elicitors.^2,3 However, some pathogens deliver virulence proteins that target host protein to overcome the plant immunity response. Most plants have evolved the corresponding resistance (R) protein to recognize effector activity, which will trigger plant resistance through effector-triggered immunity (ETI).⁴ Natural selection drives evolution of new pathogen effector proteins and plant R proteins. This tug-of-war between plants and pathogens is represented as a zig-zag-zig model.^5–7 Both PTI and ETI cause stomatal closure and hypersensitive response (HR), a programmed host cell death (PCD) to limit pathogen development.^5,8 In plants, HR is caused by proteases with caspase activity. At least eight caspase activities have now been measured in plant extracts, which were found using caspase substrates, and various caspase inhibitors can block many forms of plant programmed cell death.⁹In the past several years, vacuolar-processing enzyme (VPE) has been determined to play important roles in plant immunity responses. In this review paper, I describe the current view on the role of VPE in plant immunity, based on our own research and recent developments in this field.