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.     

Plant defenses against parasitic plants show similarities to those induced by herbivores and pathogens

Herbivores and pathogens come quickly to mind when one thinks of the biotic challenges faced by plants. Important but less appreciated enemies are parasitic plants, which can have important consequences for the fitness and survival of their hosts. Our knowledge of plant perception, signaling and response to herbivores and pathogens has expanded rapidly in recent years, but information is generally lacking for parasitic species. In a recent paper we reported that some of the same defense responses induced by herbivores and pathogens—notably increases in jasmonic acid (JA), salicylic acid (SA), and a hypersensitive-like response (HLR)—also occur in tomato plants upon attack by the parasitic plant Cuscuta pentagona (field dodder). Parasitism induced a distinct pattern of JA and SA accumulation, and growth trials using genetically-altered tomato hosts suggested that both JA and SA govern effective defenses against the parasite, though the extent of the response varied with host plant age. Here we discuss similarities between the induced responses we observed in response to Cuscuta parasitism to those previously described for herbivores and pathogens and present new data showing that trichomes should be added to the list of plant defenses that act against multiple enemies and across kingdoms.Key words: Cuscuta, induced defenses, parasitic plant, jasmonic acid, salicylic acid, phytohormones, hypersensitive response, trichomes, defense signalingSeveral thousand species of plants are parasitic, stealing water and nutrients from other plants through a specialized feeding structure, the haustorium.¹ Haustoria are thought to be modified roots that grow into tissues and fuse with the vascular system of their photosynthetic hosts.¹ Considering that these parasites include some of the world''s most devastating agricultural pests² and are influential, fascinating components of natural communities,^1,3 surprisingly little is known about host defenses induced by parasitic plants. To address this shortcoming, we used a metabolomics approach to track biochemical changes induced in tomato shoots by invasion of C. pentagona haustoria.⁴We found that parasitism induced large increases in both JA and SA beginning about 24 hr after formation of haustoria began, but that production of JA and SA was largely separated in time. Host production of JA was transitory and reached a maximum at 36 hr, whereas SA peaked 12 hr later and remained elevated 5 d later. We also found that C. pentagona grew larger on mutant tomato plants in which the SA (NahG) or JA (jasmonic acid-insensitive1) pathways were disrupted, suggesting that these hormones can act independently to reduce parasite growth. Taken together, these findings suggest the staggered production of JA and SA may be an adaptive response to parasitism—by sequentially activating the JA and SA pathways, tomato plants may minimize the potential for cross-talk between these sometimes antagonistic pathways^5,6 and utilize both signaling molecules.^6,7 Thus, defenses against C. pentagona contain elements characteristic of responses to both herbivores (primarily JA-mediated⁸) and pathogens (primarily SA-mediated⁹)—though it should be noted that some herbivores induce SA¹⁰ and some pathogens JA.¹¹ It is worth noting that parasitism induced predominately cis-JA, the same jasmonate isomer induced by herbivore feeding.¹² Host responses to Cuscuta seem to most resemble that of known plant responses to some pathogens in which a similar sequence of JA and SA production is required to limit disease.¹³C. pentagona also triggered a hypersensitive-like response (HLR) localized around the points of parasite attachment. Using a trypan blue staining technique, we verified host cell death in these parasite-induced lesions. The deposition of eggs by some insect herbivores can elicit the formation of necrotic tissue,¹⁴ but localized cell death is most widely associated with the hypersensitive response (HR) of plants to pathogens. This complex early defense response can restrict the growth and spread of viruses, fungi and bacteria.⁹ Our work adds to existing evidence¹⁵ that the Cuscuta-induced HLR can play a similar role by preventing or limiting the growth of the parasite.An interesting discovery was that the first attachment by C. pentagona elicited almost no response from young 10-day-old hosts, whereas a subsequent attachment after 10 days induced the wholesale changes discussed above (we also found changes in abscisic acid and free fatty acids). Trials in which we varied the age of the host and parasite indicated that host age, rather than a priming effect on defenses, determined the magnitude of response. We have previously observed that Cuscuta spp. in natural populations germinate very early in the growing season, and hypothesized that this tactic promotes successful parasitism by ensuring the presence of young hosts; recent field work seems to corroborate this.¹⁶ As with the response to Cuscuta parasitism, levels of host plant defenses against insects¹⁷ and pathogens¹⁸ are known to be vary with host age.In an earlier paper we reported that tomato plants parasitized by C. pentagona released greater amounts of volatiles than did unparasitized control plants.¹⁹ The production and release of volatiles is a hallmark of plant responses to feeding by herbivores.²⁰ Herbivore-induced volatiles serve as an indirect plant defense by attracting herbivores'' natural enemies,²¹ repelling herbivores,²² or acting as intra-plant signals that prime systemic responses.²³ Although less well documented, pathogen attack can also induce emissions of volatile compounds,²⁴ some of which are antimicrobial and may serve as a direct defense against infection.²⁵ The same volatile compounds induced by Cuscuta (e.g., 2-carene, α-pinene, limonene, β-phellandrene) were also induced by caterpillar feeding and application of JA.¹⁹ Like herbivores, the JA induced by C. pentagona may regulate the emissions of plant volatiles. Whether or how parasitic plant-induced volatiles might function in defense is unknown, but they presumably could affect host plant choice by Cuscuta seedlings, which use plant volatiles to locate and select hosts.²⁶Following on from our previous studies we examined the potential role of host trichomes in resistance to parasitism by C. pentagona. Plant trichomes have been long appreciated as the first line of defense against insect herbivores^27,28 and more recently pathogens.²⁹ We hypothesized that trichomes could also defend against parasitic plants based on our observations that (1) tomato trichomes become denser with age (Fig. 1), notably on hypocotyls which is the first area contacted by Cuscuta seedlings, and (2) these trichomes can act as a physical barrier to C. pentagona seedlings. To test this hypothesis we allowed seedlings of C. pentagona to attach to 25-day-old tomato plants (Solanum lycopersicum ‘Halley 3155’) in a climate controlled growth chamber. Of 20 trials conducted, in six (30%) the parasite seedling was completely blocked by trichomes and was unable to reach the host stem—the parasite perished in each of these. Type I glandular trichomes, which are several millimeters long with a glandular tip,³⁰ were primarily responsible for the blocking effect. Thus, trichomes can defend against parasitic plants in a manner analogous to herbivores by physically obstructing their movement. Interestingly, the effectiveness of trichomes is also dependent on age of the host since those on younger tomato plants (<20 days old) are too sparse to impede Cuscuta seedlings (Fig. 1).Open in a separate windowFigure 1A newly germinated Cuscuta pentagona seedling encircles and attaches to the hypocotyl of a 10-day-old tomato seedling; the early development of haustoria are visible as nod-like swellings. The trichomes on hypocotyls of young tomato seedlings are not dense enough to affect C. pentagona seedlings, but the increased density of trichomes on 25-day-old plants can act as a physical barrier that blocks parasite seedlings (inset).Considering that the majority of plant defenses are mediated by only a small number of master regulators (e.g., JA, SA, ethylene),⁷ it is not surprising that plant responses to parasitic plants share commonalities with those induced by herbivores and pathogens. These few molecules mediate complex, interacting signaling networks that can be variously activated and modified by plants to tune defenses against a seemingly endless variety of attackers.⁷ Our finding that JA and SA act to defend plants from attack by other plants, further support these phytohormones as ‘global’ defense signals. It is also apparent that constitutive defenses, such as trichomes, can be effective against diverse antagonists (e.g., herbivores and parasitic plants). These new insights into host defenses against parasitic plants suggest many avenues of needed research including the molecular events induced by parasitic plant attack, the parasite-derived cues that elicit responses, and the ways in which JA and SA act to reduce parasite growth. Finally, our findings suggest it might be possible to manipulate induced responses or host plant age by varying planting date to control parasitic plants in agriculture.     

Neutralization of Clostridium difficile toxin A using antibody combinations

The pathogenicity of Clostridium difficile (C. difficile) is mediated by the release of two toxins, A and B. Both toxins contain large clusters of repeats known as cell wall binding (CWB) domains responsible for binding epithelial cell surfaces. Several murine monoclonal antibodies were generated against the CWB domain of toxin A and screened for their ability to neutralize the toxin individually and in combination. Three antibodies capable of neutralizing toxin A all recognized multiple sites on toxin A, suggesting that the extent of surface coverage may contribute to neutralization. Combination of two noncompeting antibodies, denoted 3358 and 3359, enhanced toxin A neutralization over saturating levels of single antibodies. Antibody 3358 increased the level of detectable CWB domain on the surface of cells, while 3359 inhibited CWB domain cell surface association. These results suggest that antibody combinations that cover a broader epitope space on the CWB repeat domains of toxin A (and potentially toxin B) and utilize multiple mechanisms to reduce toxin internalization may provide enhanced protection against C. difficile-associated diarrhea.Key words: Clostridium difficile, toxin neutralization, therapeutic antibody, cell wall binding domains, repeat proteins, CROPs, mAb combinationThe most common cause of nosocomial antibiotic-associated diarrhea is the gram-positive, spore-forming anaerobic bacillus Clostridium difficile (C. difficile). Infection can be asymptomatic or lead to acute diarrhea, colitis, and in severe instances, pseudomembranous colitis and toxic megacolon.^1,2The pathological effects of C. difficile have long been linked to two secreted toxins, A and B.^3,4 Some strains, particularly the virulent and antibiotic-resistant strain 027 with toxinotype III, also produce a binary toxin whose significance in the pathogenicity and severity of disease is still unclear.⁵ Early studies including in vitro cell-killing assays and ex vivo models indicated that toxin A is more toxigenic than toxin B; however, recent gene manipulation studies and the emergence of virulent C. difficile strains that do not express significant levels of toxin A (termed “A⁻ B⁺”) suggest a critical role for toxin B in pathogenicity.^6,7Toxins A and B are large multidomain proteins with high homology to one another. The N-terminal region of both toxins enzymatically glucosylates small GTP binding proteins including Rho, Rac and CDC42,^8,9 leading to altered actin expression and the disruption of cytoskeletal integrity.^9,10 The C-terminal region of both toxins is composed of 20 to 30 residue repeats known as the clostridial repetitive oligopeptides (CROPs) or cell wall binding (CWB) domains due to their homology to the repeats of Streptococcus pneumoniae LytA,^11–14 and is responsible for cell surface recognition and endocytosis.^12,15–17C. difficile-associated diarrhea is often, but not always, induced by antibiotic clearance of the normal intestinal flora followed by mucosal C. difficile colonization resulting from preexisting antibiotic resistant C. difficile or concomitant exposure to C. difficile spores, particularly in hospitals. Treatments for C. difficile include administration of metronidazole or vancomycin.^2,18 These agents are effective; however, approximately 20% of patients relapse. Resistance of C. difficile to these antibiotics is also an emerging issue^19,20 and various non-antibiotic treatments are under investigation.^20–25Because hospital patients who contract C. difficile and remain asymptomatic have generally mounted strong antibody responses to the toxins,^26,27 active or passive immunization approaches are considered hopeful avenues of treatment for the disease. Toxins A and B have been the primary targets for immunization approaches.^20,28–33 Polyclonal antibodies against toxins A and B, particularly those that recognize the CWB domains, have been shown to effectively neutralize the toxins and inhibit morbidity in rodent infection models.³¹ Monoclonal antibodies (mAbs) against the CWB domains of the toxins have also demonstrated neutralizing capabilities; however, their activity in cell-based assays is significantly weaker than that observed for polyclonal antibody mixtures.^33–36We investigated the possibility of creating a cocktail of two or more neutralizing mAbs that target the CWB domain of toxin A with the goal of synthetically re-creating the superior neutralization properties of polyclonal antibody mixtures. Using the entire CWB domain of toxin A, antibodies were raised in rodents and screened for their ability to neutralize toxin A in a cell-based assay. Two mAbs, 3358 and 3359, that (1) both independently demonstrated marginal neutralization behavior and (2) did not cross-block one another from binding toxin A were identified. We report here that 3358 and 3359 use differing mechanisms to modify CWB-domain association with CHO cell surfaces and combine favorably to reduce toxin A-mediated cell lysis.     

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     

Hypoxia: A novel function for VIN3

VERNALIZATION INSENSITIVE 3 (VIN3) encodes a PHD domain chromatin remodelling protein that is induced in response to cold and is required for the establishment of the vernalization response in Arabidopsis thaliana.¹ Vernalization is the acquisition of the competence to flower after exposure to prolonged low temperatures, which in Arabidopsis is associated with the epigenetic repression of the floral repressor FLOWERING LOCUS C (FLC).^2,3 During vernalization VIN3 binds to the chromatin of the FLC locus,¹ and interacts with conserved components of Polycomb-group Repressive Complex 2 (PRC2).^4,5 This complex catalyses the tri-methylation of histone H3 lysine 27 (H3K27me3),^4,6,7 a repressive chromatin mark that increases at the FLC locus as a result of vernalization.^4,7–10 In our recent paper¹¹ we found that VIN3 is also induced by hypoxic conditions, and as is the case with low temperatures, induction occurs in a quantitative manner. Our experiments indicated that VIN3 is required for the survival of Arabidopsis seedlings exposed to low oxygen conditions. We suggested that the function of VIN3 during low oxygen conditions is likely to involve the mediation of chromatin modifications at certain loci that help the survival of Arabidopsis in response to prolonged hypoxia. Here we discuss the implications of our observations and hypotheses in terms of epigenetic mechanisms controlling gene regulation in response to hypoxia.Key words: arabidopsis, VIN3, FLC, hypoxia, vernalization, chromatin remodelling, survival     

LeEix1 functions as a decoy receptor to attenuate LeEix2 signaling

The receptors for the fungal elicitor EIX (LeEix1 and LeEix2) belong to a class of leucine-rich repeat cell-surface glycoproteins with a signal for receptor-mediated endocytosis. Both receptors are able to bind the EIX elicitor while only the LeEix2 receptor mediates defense responses. We show that LeEix1 acts as a decoy receptor and attenuates EIX induced internalization and signaling of the LeEix2 receptor. We demonstrate that BAK1 binds LeEix1 but not LeEix2. In plants where BAK1 was silenced, LeEix1 was no longer able to attenuate plant responses to EIX, indicating that BAK1 is required for this attenuation. We suggest that LeEix1 functions as a decoy receptor for LeEix2, a function which requires the kinase activity of BAK1.Key words: LRR-RLP, LeEix, Bak1, decoy receptor, endocytosisLeucine-rich-repeat receptor proteins (LRR-RLPs) have been linked with defense response signaling in plants.^1–5 The tomato Cf genes which mediate resistance to Cladosporium fulvum encode LRR-RLPs. Additional LRR-RLPs include the tomato Verticillium (Ve) resistant proteins^6,7 and the LeEix proteins.⁸ The Eix receptors (LeEix1 and LeEix2) contain a signal for receptor-mediated endocytosis, which we have previously shown to be essential for proper induction of defense responses.^9,10 Both receptors are able to bind Eix, but only LeEix2 mediates EIX-induced defense.⁸ In a recent work we demonstrate that LeEix1 attenuates Eix-induced internalization and signaling, and heterodimerizes with LeEix2 upon application of Eix.¹¹ Our work further shows that the brassinosteroid co-receptor Bri-Associated Kinase 1 (BAK1) binds LeEix1 but not LeEix2. In BAK1-silenced plants, LeEix1 was no longer able to attenuate plant responses to Eix, indicating that BAK1 is required for this attenuation and leading to the hypothesis that LeEix1 functions as a decoy receptor for LeEix2.¹¹     

Plant defensins: Defense,development and application

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

Loss of LORELEI function in the pistil delays initiation but does not affect embryo development in Arabidopsis thaliana

Double fertilization, uniquely observed in plants, requires successful sperm cell delivery by the pollen tube to the female gametophyte, followed by migration, recognition and fusion of the two sperm cells with two female gametic cells. The female gametophyte not only regulates these steps but also controls the subsequent initiation of seed development. Previously, we reported that loss of LORELEI, which encodes a putative glycosylphosphatidylinositol (GPI)-anchored protein, in the female reproductive tissues causes a delay in initiation of seed development. From these studies, however, it was unclear if embryos derived from fertilization of lre-5 gametophytes continued to lag behind wild-type during seed development. Additionally, it was not determined if the delay in initiation of seed development had any lingering effects during seed germination. Finally, it was not known if loss of LORELEI function affects seedling development given that LORELEI is expressed in eight-day-old seedlings. Here, we showed that despite a delay in initiation, lre-5/lre-5 embryos recover, becoming equivalent to the developing wild-type embryos beginning at 72 hours after pollination. Additionally, lre-5/lre-5 seed germination, and seedling and root development are indistinguishable from wild-type indicating that loss of LORELEI is tolerated, at least under standard growth conditions, in vegetative tissues.Key words: LORELEI, glycosylphosphatidylinositol (GPI)-anchored protein, embryogenesis, DD45, seed germination, primary and lateral root growth, seedling developmentDouble fertilization is unique to flowering plants. Upon female gametophyte reception of a pollen tube, the egg and central cells of the female gametophyte fuse with the two released sperm cells to form zygote and endosperm, respectively and initiate seed development.¹ The female gametophyte controls seed development by (1) repressing premature central cell or egg cell proliferation until double fertilization is completed,^1–3 (2) supplying factors that mediate early stages of embryo and endosperm development^1,4,5 and (3) regulating imprinting of genes required for seed development.^1,6The molecular mechanisms underlying female gametophyte control of early seed development are poorly understood. Although much progress has been made in identifying genes and mechanisms by which the female gametophyte represses premature central cell or egg cell proliferation until double fertilization is completed and regulates imprinting of genes required for seed development,^1,6 only a handful of female gametophyte-expressed genes that affect early embryo^7,8 and endosperm⁹ development after fertilization have been characterized. This is particularly important given that a large number of female gametophyte-expressed genes likely regulate early seed development.⁵We recently reported on a mutant screen for plants with reduced fertility and identification of a mutant that contained a large number of undeveloped ovules and very few viable seeds.¹⁰ TAIL-PCR revealed that this mutant is a new allele of LORELEI(LRE) [At4g26466].^10,11 Four lre alleles have been reported;¹¹ so, this mutant was designated lre-5.¹⁰ The Arabidopsis LORELEI protein contains 165 amino acids and possesses sequence features indicative of a glycosylphosphatidylinositol (GPI)-anchor containing cell surface protein. GPI-anchors serve as an alternative to transmembrane domains for anchoring proteins in cell membranes and GPI-anchored proteins participate in many functions including cell-cell signaling.¹²     

Plant encoded 1-aminocyclopropane-1-carboxylic acid deaminase activity implicated in different aspects of plant development

Proper plant development is dependent on the coordination and tight control of a wide variety of different signals. In the study of the plant hormone ethylene, control of the immediate biosynthetic precursor 1-aminocyclopropane-1-carboxylic acid (ACC) is of interest as the level of ethylene can either help or hinder plant growth during times of stress. It is known that ACC can be reversibly removed from the biosynthesis pathway through conjugation into other compounds. We recently reported that plants can also irreversibly remove ACC from ethylene production through the activity of a plant encoded ACC deaminase. Heretofore only found in bacteria, we showed that there was ACC deaminase activity in both Arabidopsis and in developing wood of poplar. Here we extend this original work and show that there is also ACC deaminase activity in tomato plants, and that this activity is regulated during tomato fruit development. Further, using an antisense construct of AtACD1 in Arabidopsis, we investigate the role of ACC deamination during salt stress. Together these studies shed light on a new level of control during ethylene production in a wide variety of plant species and during different plant developmental stages.Key words: tomato fruit ripening, wood development, stress response, hormone, antisense, synthesisHormones are a class of signaling molecules produced and sensed at very low levels; therefore control of their biosynthesis is crucial for proper plant development. The plant hormone ethylene has been studied for over a century and can positively impact plant development, such as in the initiation of fruit ripening, but ethylene accumulation can also induce widespread damage during stress responses.¹ Ethylene is produced in two steps from the S-adenosylmethionine (SAM) that is derived from the Yang cycle.² In the first committed step, SAM is converted into 1-aminocyclopropane-1-carboxcylic acid (ACC) via the action of ACC SYNTHASEs (ACSs).³ ACC is then converted into ethylene by ACC OXIDASEs (ACOs), a particular adaptation of flowering plants.⁴ Once ACC is produced, there are few proven pathways that can divert it from conversion into ethylene. ACC can be conjugated into malonyl-1-aminocyclopropane- 1-carboxylic acid (MACC) through the activity of ACC malonyl transferase⁵ or to 1-(γ-L-glutamyl-amino) cyclopropane-1-carboxylic acid (GACC) via γ-glutamyltranspeptidase.⁶ In bacteria, another pathway exists that can break down ACC obtained from plants through an irreversible deamination process.⁷ Through heterologous expression of bacterial ACC DEAMINASEs (ACDs) in plants it has been possible to engineer plants that have reduced production of ethylene by affecting the native pools of ACC.⁸ Until recently no ACC deaminase pathway has ever been proven in plants, although a number of different plant genomes encode genes which bear sequence homology to bacterial ACDs. Should these genes code for active ACDs, this would provide an additional level of control for ethylene production beyond the activity of ACSs and ACOs. Recently we reported that Arabidopsis and Populus have inherent ACC deaminase activity, and we showed that this activity in Arabidopsis is due, in part, to the product of ACC DEAMINASE1 (AtACD1) (At1g48420).⁹ This discovery raises many questions concerning the role of ACC deaminases during ethylene mediated processes in a number of different plant models. We report here some of our preliminary findings in the areas of tomato fruit ripening and salt stress in Arabidopsis.As precise control of ethylene levels is essential during climacteric fruit development, in parallel with our reported studies we also studied ACC deaminase activity in developing tomato fruit. Ethylene production during ripening in tomato is controlled by ethylene receptor turnover¹⁰ and conjugation of ACC by MACC and GACC.^6,11,12 We found that tomatoes also have inherent ACD activity, and that this activity varies over ripening of the fruit (Fig. 1; solid line). During the immature green stage in tomato development ACC deaminase activity was low. This activity increased significantly during the ‘late breaker’ stage, just prior to the orange/red stage of development, and then decreased during later stages of tomato ripening. Also shown in this figure are the predicted levels of ethylene during fruit development. It is interesting to note that the highest amount of ACC deaminase activity coincides with the drop in ethylene levels soon after the breaker stage (Fig. 1; dashed line; based on Brady¹³). Our data would suggest that, in addition to ethylene receptor turnover and GACC and MACC activity, ACC deaminase activity may also help control ethylene levels. It has already been shown that constitutive expression of a bacterial ACC deaminase in tomato can delay the rate of tomato fruit ripening by reduction of ethylene production.⁸ Although ACD activity is evident during ripening in tomato, the gene responsible has not been identified. Recently a tomato gene with sequence similarity to bacterial ACC deaminases was tested for ACD activity. It was found that, despite the close sequence similarity, this gene (accession number {"type":"entrez-nucleotide","attrs":{"text":"EU639448","term_id":"186920268","term_text":"EU639448"}}EU639448) did not have ACD activity.¹⁴ Therefore, additional work must be done to isolate the gene responsible for the ACD activity we demonstrate in tomato fruit.Open in a separate windowFigure 1Tomato fruits exhibit AC deaminase activity during ripening. A plot of ACC Deaminase activity (Solid Line) with known levels of ethylene production during ripening (Dashed Line; Brady¹³) superimposed over pictures of the corresponding stage of tomato development. *Indicates significant increase in activity (†nmol mg⁻¹ hr⁻¹). AC deaminase activity analysis was performed on total tomato fruit protein as per Penrose and Glick (2003).²¹Our discovery of a plant encoded ACC deaminase in Arabidopsis allows us, for the first time, to downregulate ACC deaminase activity and investigate how this affects plant development. Previously, we showed that downregulation of AtACD1 using antisense resulted in up to a 30% reduction in ACD activity and up to a 2.5-fold increase in the evolution of ethylene.⁹ We showed that this difference in ACD activity was sufficient to alter hypocotyl elongation during Arabidopsis germination on different concentrations of ACC. It was unknown, however, if this difference was sufficient to affect other areas of development, such as stress response, in Arabidopsis. The expression of bacterial ACC deaminases in plants are known to increase plant resistance to a number of stressors due to decreased ethylene evolution.^15–18 Based on microarray data, it is known that AtACD1 expression is upregulated 150% during salt stress¹⁹ and functionally it has been demonstrated that ACC production is increased in salt stressed roots²⁰ and overexpression of bacterial ACDs in canola increases salt tolerance.¹⁸ It was unknown, however, if a reduction in native ACD activity would result in reduced vigour of plants grown on increasing concentrations of sodium chloride. We observed that there was no significant difference in rosette size, leaf production or percent dry weight between wildtype and three independent Arabidopsis lines expressing the AtACD1 antisense construct when grown on MS media without salt (Fig. 2A–C). As the concentration of salt increased in the growth media it was found that the antisense lines also did not differ from wildtype in their growth. The lack of a definitive phenotype under salt stress may mean that the level of reduced ACD activity achieved in the AtACD1 antisense lines was not sufficient to quantifiably affect the development of Arabidopsis. Additionally, as ethylene is not the only factor that affects a plant’s survival during times of salt stress, it is also possible that the plants were able to compensate for increased ethylene production in the AtACD1 antisense lines to promote normal plant development. This finding highlights the complex nature of the different signals involved in a plant’s response to salt stress and the need for a better understanding of the role of plant ACDs and how the plant may compensate for altered ACD activity.Open in a separate windowFigure 2Growth and development of Arabidopsis wildtype and three Antisense AtACD1 lines on increasing concentrations of salt. Stratified wildtype Arabidopsis (Col-0) and three independent transgenic lines expressing an antisense construct of AtACD1 (A1, A2, A3) were sown on 0 mM NaCl (Dark Grey Bars), 100 mM NaCl (White Bars), 125 mM NaCl (Black Bars) and 150 mM NaCl (Light Grey Bars) and allowed to germinate and grow for 2 weeks under long-day conditions (16 h light/8 h dark) at a light intensity of 130 to 190 µE m-2s⁻¹ at the rosette level at 21°C in Econair AC -60 growth chambers. Plants were analyzed for rosette diameter (A), leaf production (B) and percent dry weight (C). Error bars are ± SE.In the known framework of ethylene synthesis our work has shown that plants do have the ability to reduce ethylene synthesis by irreversibly deaminating ACC through the action of a native ACC deaminase. Further to our first study, we show here that there is inherent ACC deaminase activity in tomatoes and that this activity varies during tomato ripening in a manner consistent with a factor that is involved in the regulation of ethylene levels. We also show here that transgenic Arabidopsis lines with a mild reduction in ACD1 activity do not have an obvious affect on mediation of salt stress. This finding, however, does not preclude a role for ACD1 in mediating other aspects of plant development or in affecting plant development during other types of plant stress (i.e., drought). Therefore, there still remain many questions to answer concerning the role of plant encoded ACC deaminases and many exciting avenues of ethylene regulation to pursue. The identification and exploitation of tomato, poplar and other plant ACC deaminases could be used to alter fruit ripening, wood production and stress tolerance—all aspects of plant development that are economically and scientifically important.     

Strigolactones: Internal and external signals in plant symbioses?

As the newest plant hormone, strigolactone research is undergoing an exciting expansion. In less than five years, roles for strigolactones have been defined in shoot branching, secondary growth, root growth and nodulation, to add to the growing understanding of their role in arbuscular mycorrhizae and parasitic weed interactions.¹ Strigolactones are particularly fascinating as signaling molecules as they can act both inside the plant as an endogenous hormone and in the soil as a rhizosphere signal.^2-4 Our recent research has highlighted such a dual role for strigolactones, potentially acting as both an endogenous and exogenous signal for arbuscular mycorrhizal development.⁵ There is also significant interest in examining strigolactones as putative regulators of responses to environmental stimuli, especially the response to nutrient availability, given the strong regulation of strigolactone production by nitrate and phosphate observed in many species.^5,6 In particular, the potential for strigolactones to mediate the ecologically important response of mycorrhizal colonization to phosphate has been widely discussed. However, using a mutant approach we found that strigolactones are not essential for phosphate regulation of mycorrhizal colonization or nodulation.⁵ This is consistent with the relatively mild impairment of phosphate control of seedling root growth observed in Arabidopsis strigolactone mutants.⁷ This contrasts with the major role for strigolactones in phosphate control of shoot branching of rice and Arabidopsis^8,9 and indicates that the integration of strigolactones into our understanding of nutrient response will be complex. New data presented here, along with the recent discovery of phosphate specific CLE peptides,¹⁰ indicates a potential role for PsNARK, a component of the autoregulation of nodulation pathway, in phosphate control of nodulation.     

Nitric oxide (NO)-dependent and NO-independent signaling pathways act in ABA-inhibition of stomatal opening

We investigated the role of nitric oxide (NO) in ABA-inhibition of stomatal opening in Vicia faba L. in different size dishes. When a large dish (9 cm diameter) was used, ABA induced NO synthesis and the NO scavenger reduced ABA-inhibition of stomatal opening. When a small dish (6 cm diameter) was used, ABA induced stomatal closure and inhibited stomatal opening. The NO scavenger was able to reduce ABA-induced stomatal closure, but unable to reverse ABA-inhibition of stomatal opening. Furthermore, NO was not synthesized in response to ABA, indicating that NO is not required for ABA-inhibition of stomatal opening in the small dish. These results indicated that an NO-dependent and an NO-independent signaling pathway participate in ABA signaling pathway. An NO-dependent pathway is the major player in ABA-induced stomatal closure. However, in ABA-inhibition of stomatal opening, an NO-dependent and an NO-independent pathway act: different signaling molecules participate in ABA-signaling cascade under different environmental condition.Key words: ABA, environmental condition, nitric oxide, stomata, Vicia faba LNitric oxide (NO) is a key signaling molecule in plants.^1,2 It functions in disease resistance and programmed cell death,^3,4 root development,^5,6 and plant responses to various abiotic stresses.^1,2,7,8 In addition, NO is required for stomatal closure in response to ABA in several species including Arabidopsis, Vicia faba, pea, tomato, barley, and wheat.^9–11 ABA-inhibition of stomatal opening is a distinct process from ABA-induced stomatal closure.^12,13 In V. faba, these two processes employ a similar signaling pathway; NO is also a second messenger molecule for ABA-inhibition of stomatal opening in a large dish.¹⁴ In this study, we examined the role of NO in ABA-inhibition of stomatal opening using different dish sizes. In a small dish, NO is not involved in ABA-inhibition of stomatal opening: the NO-independent signaling pathway is the major player in it.