The importance of floral signals in the establishment of plant-ant mutualisms

Visual and olfactory floral signals are essential for the establishment of plant-pollinator mutualisms. Different batteries of floral features attract different pollinators and may achieve specific relationships that are essential for the immediate plant reproductive success, and at an evolutionary time scale have been of vital importance in the radiation of Angiosperms. We have found that mutualistic services by ants, insects traditionally considered ineffective pollinators, are essential for the pollination of Cytinus hypocistis (Cytinaceae), a Mediterranean root holoparasitic plant. Diverse floral signals, mainly nectar characteristics and floral scent could be playing a key role in the attraction of different species of ants, which pollinate effectively the flowers. Surprisingly, the abundance of other insects foraging in this parasite was very low and, although this scarcity could be due in part to the presence of ants, we suggest that different floral features exhibited by C. hypocistis could be evolving for attracting ants. Based on some current findings, we suspect that the study of floral signals in Cytinaceae is critical in the understanding the divergence of pollination systems in this fascinating family of parasitic plants.Key words: ant, Cytinus hypocistis, Cytinaceae, floral signal, nectar, plant-animal interaction, scentThe flowers of angiosperms exhibit an amazing variety of floral and nectar colors,^1,2 floral morphology and displays,^3,4 floral scents⁵ and nectar characteristics,^6,7 that may influence pollinator type and pollination quality. The existence of those signals help to the establishment of interactions among plants and pollinators, that range from drastic generalists, when flowers are visited by an elevated number of pollinators, to extreme specialists, being the plant pollinated by only one or a few species of pollinators.^8,9In the majority of terrestrial ecosystems, ants stand out as one of the most common floral visitors.¹⁰ In spite of such ubiquity, ants have been largely considered as ineffective pollinators, mainly due to their small size, erratic behaviour and the presence of metapleural glands that produce antibiotic secretions reducing pollen viability.^11,12 Moreover, it has been postulated that ants act as nectar thieves and reduce visitation by other potential pollinator.¹⁰ However, new findings are highlighting that their role on pollination is not fully understood. Recently we have described, combining a four-year field observation study with experimental pollination treatments at six study sites, a new ant-plant mutualism, between the holoparasitic plant Cytinus hypocistis (Cytinaceae) and different ant species,¹³ joining a growing body of evidence stressing the prominent role that ants are playing in some plant-pollinator systems.^14–17 Nevertheless, in contrast to preceding studies where only one ant species pollinated the plants (but see refs. ¹⁷ and ¹⁸) in C. hypocistis as many as ten ant species behave as true pollinators. Moreover, our study has shown that the different ant species that pollinate C. hypocistis differ in their effectiveness as pollinators, as commonly observed in other pollinator guilds.^19,20 We therefore suggest that generalizations about the importance and quality of ants as pollinators should be avoided or made with caution unless backed by careful field measurements.Our study system was a monoecious rootless, stemless and leafless holoparasitic plant with inflorescences appearing at ground level. Although C. hypocistis does not exhibit typical features of the purported ‘ant-pollination syndrome’,²¹ a number of floral signals could be playing key roles in the establishment of the plant-ant interactions. Among them, the low stature of plants that make flowers to stay a ground level, in combination with sweet scented flowers that offer concentrated nectar and large quantity of pollen, are signals that may enhance the attractiveness of C. hypocistis to diurnal and nocturnal ants that pollinate flowers efficiently. Flying insect visits were unexpectedly low, and only a fly was a predictable visitor. The presence of ants can discourage flying pollinators from visiting the flowers,²² but we suspect that this low number of visits can be explained by the floral features exhibited by C. hypocistis which, although probably still no specialized, may be becoming more important for attracting ants.It has been postulated that different species of animals are attracted by diverse sets of visual (colour and floral shape) and chemical (floral scent, nectar features) floral characteristics.^5,6,9 These features are critical for plant reproductive success,^23,24 and at an evolutionary time scale they have been of vital importance in the radiation of Angiosperms.²⁵ Although our study does not specifically deal with the relative importance of different floral signals, circumstantial evidence suggests that in the family Cytinaceae various signalling-related features could be essential for the establishment of plant-pollinator mutualisms. More specifically, floral scent seems to be an important channel of communication between these flowering plants and their pollinators and has been a likely key factor in the evolution of pollination mechanisms in this outstanding family of parasitic plants. In the only three studies conducted on plant-pollinator systems in the family Cytinaceae a remarkable diversity of mutualisms has been observed. First, in Mexico, the yeasty and unpleasant scent produced by the dark flowers of Bdallophyton bambusarum has been described to attract carrion flies,²⁶ a common pollinator guild in other parasitic plants such as Rafflesiaceae or Hydnoraceae, with a characteristic pollination system by deceit with flowers mimicking insect mating or egg-laying habitats.²⁷ Second, in South Africa, the crimson flowers of Cytinus visseri emit a strong scent dominated by aliphatic compounds that play a central role in attracting small mammals, the only flower visitors and pollinators of this species.²⁸ And third, our recent findings on C. hypocistis in Spain and Morocco point to an important effect of floral scent in attracting ants, its main pollinators. Although research on the importance of floral signals to attract pollinators in this family is still in its infancy, recent findings make of Cytinaceae an ideal model to study at an intrafamily level the influence of divergent selection from pollinators on the evolution of high floral signal diversity. Mechanisms acting on plant-animals signalling obviously deserve further studies to provide an accurate picture of the importance of visual and chemical cues on the evolution of pollination systems in Angiosperms.     

Pseudomyrmex ants and Acacia host plants join efforts to protect their mutualism from microbial threats

Plants express numerous ‘pathogenesis-related’ (PR) proteins to defend themselves against pathogen infection. We recently discovered that PR-proteins such as chitinases, glucanases, peroxidases and thaumatin-like proteins are also functioning in the protection of extra-floral nectar (EFN) of Mexican Acacia myrmecophytes. These plants produce EFN, cellular food bodies and nesting space to house defending ant species of the genus Pseudomyrmex. More than 50 PR-proteins were discovered in this EFN and bioassays demonstrated that they actively can inhibit the growth of fungi and other phytopathogens. Although the plants can, thus, express PR-proteins and secrete them into the nectar, the leaves of these plants exhibit reduced activities of chitinases as compared to non-myrmecophytic plants and their antimicrobial protection depends on the mutualistic ants. When we deprived plants of their resident ants we observed higher microbial loads in the leaves and even in the tissue of the nectaries, as compared to plants that were inhabited by ants. The indirect defence that is achieved through an ant-plant mutualism can protect plants also from infections. Future studies will have to investigate the chemical nature of this mechanism in order to understand why plants depend on ants for their antimicrobial defence.Key words: ant-plant mutualism, indirect defense, pathogen resistance, pathogenesis-related proteinPlants fall victim to multiple attackers from various kingdoms and have evolved numerous strategies to defend themselves against herbivores and microbial pathogens. Pathogen resistance is usually based on particular cell wall properties, secondary compounds (phytoalexins) and on pathogenesis-related (PR) proteins.^1,2 Plant resistance to pathogens is thus, mainly based on plant traits that interact with the pathogen and therewith functions as a “direct” defence mechanism. Resistance to herbivores, by contrast, can also be “indirect”: plants house or attract carnivores, which fulfil the defensive function.³ Defensive ant-plant interactions are common mutualisms in which plants provide to ants an array of rewards that comprises both food rewards and domatia (nesting space).^3,4 Ants are attracted or nourished by plant-derived food rewards and defend the plants against herbivores.^3,5 In obligate interactions, myrmecophytes are inhabited by specialised ants during major parts of their life⁴ and the ants are entirely dependent on the food rewards and nesting space that are provided by their host. These ants, in return, defend their host efficiently and aggressively against herbivores and encroaching vegetation. Only recently we found that this protective service might also cover the defence against phytopathogens.In the Mexican Acacia-Pseudomyrmex mutualism, extrafloral nectar (EFN) represents an important plant trait to nourish symbiotic ant colonies. EFN is secreted by special glands called extrafloral nectaries and is rich in mono- and disaccharides and amino acids.⁶ Due to its high content in primary metabolites, EFN appears also attractive to exploiters, which make use of the nectar resource without providing a service to the plant.⁷ For example, EFN can serve as a suitable medium for pathogen growth and is highly prone to microbial infestations, which can have negative consequences on nectar composition.^8,9We recently found that Acacia EFN is biochemically protected from microbial infections.^10,11 More than 50 proteins could be distinguished in EFN of the myrmecophytes, A. hindsii, A. cornigera and A. collinsii, and most of these proteins were annotated as PR-proteins such as chitinases, β-1,3 glucanases, thaumatin-like proteins and peroxidases. In fact, chitinases and glucanases represented more than 50% of the total amount of proteins in EFN of myrmecophytic Acacia plants.^10,11 This dominance of PR-proteins clearly distinguishes the anti-microbial strategy of Acacia-EFN from the strategy of floral nectar of ornamental tobacco. The protection of the latter nectar is mainly based on small metabolites, such as hydrogen peroxide, which are produced by five proteins forming the ‘nectar redox cycle’.^12–14 By contrast, PR-proteins have also been reported from pollination droplets of gymnosperms,¹⁵ although their biological functions remain to be studied. For the Acacia EFN, biotests demonstrated that the chitinases and glucanases are active and that EFN successfully can inhibit the growth of various fungi and oomycetes.¹¹Nevertheless, and although the microorganisms used in these assays were phytopathogens, we have now observed that the protection from microbial infection of the Acacia plant itself depends on certain characteristics of its ant inhabitants. The occurrence of fungi and bacteria in EFN and in the tissue of leaves and nectaries was investigated under natural growing conditions. We collected samples of A. cornigera and A. hindsii plants, which were inhabited by ants or which had been deprived of ants experimentally two weeks before. The tissues were extracted and analyzed for microbial infection by cultivating the extracts on malt agar plates, in order to quantify the abundances of life fungi and bacteria as the numbers of colony-forming units (CFU). Leaves of two species of myrmecophytic Acacia plants showed a significant increase in their bacterial load when they were deprived of the mutualistic P. ferrugineus ants (Fig. 1A). This observation is redolent of an earlier observation made on Macaranga myrmecophytes in Malaysia: lesions of these plants could easily be infected with fungi when the mutualistic Crematogaster ants were absent, but not in the presence of the ants.¹⁶ Similarly, myrmecophytic Piper plants depend, at least in part, on their ant inhabitants to obtain an efficient protection from fungal pathogens.¹⁷ Myrmecophytic Maracanga plants possessed reduced activities of chitinases in their leaves¹⁶ and the same phenomenon was found in Acacia myrmecophytes.¹⁸ Thus, the leaves of the obligate ant-plants of both genera, Acacia and Macaranga, appear not to express PR-proteins at sufficient activities as to protect themselves from pathogens and symbiotic ants are required for this defensive function. The effect is specific for the defending ants, because no significant differences between plants with and without ants could be detected in the case of plants that were inhabited by the non-defending parasite, P. gracilis¹⁹ (Fig. 1B). Most interestingly, even the nectary tissue appears to require ant-mediated protection from phytopathogens, whereas the EFN itself does not: no fungi can usually be isolated from freshly collected nectar of Acacia myrmecophytes under field conditions¹⁰ (Fig. 2A), but the tissue of the nectaries became heavily infected when branches were kept free of the defending ants (Fig. 2B).Open in a separate windowFigure 1Effects of the presence and absence of the symbiotic ant P. ferrugineus and the parasitic ant P. gracilis on the abundance of bacteria [CFU mg dry leaf mass⁻¹] in leaf samples of A. cornigera (A) and A. hindsii (B). Significance levels are indicated: ns p > 0.05, *p < 0.05, **p < 0.01 and ***p < 0.001. (Two-factorial ANOVA applied separately for each plant species; independent variables: ant presence and ant species.)Open in a separate windowFigure 2Effects of presence and absence of the symbiotic ant P. ferrugineus on fungal abundance in EFN (A) and nectar tissue (B) of A. cornigera and A. hindsii. Significance levels are indicated: ns p > 0.05, *p < 0.05, **p < 0.01 and ***p < 0.001. (Two-factorial ANOVA; independent variables: ant presence and plant species).Our new findings highlight the joint efforts of both ant and plant that are required for an efficient protection from pathogens of the myrmecophyte host plants, and, thus, for the prevention of this mutualism from exploitation by microorganisms. It remains open why the myrmecophytic Acacia plants exhibit reduced chitinase activities in their leaves¹⁸ and thus depend on ants for their antimicrobial protection, although they are fully equipped to express functioning PR-proteins and secrete them into their EFN.^10,11 We could assume that an antimicrobial protection by ants is less costly for the plant than its own biochemical defence mechanisms. Alternatively, plant pathogens, which express multiple effectors in order to suppress their host resistance strategies,¹ might be less capable to cope with ant-derived resistance mechanisms. Every attempt of an explanation, however, remains speculative as long as we do not understand how the ants can protect the leaves of their host plant from infection. The chemical mechanisms remain to be analyzed and may comprise both an ant-mediated resistance induction in the plant and directly ant-derived antimicrobial compounds. Most importantly, the joined efforts of both plant host and ant inhabitant are required to keep leaves, nectaries and nectar free of microbial infections and microbial pathogens have been identified as a further target of the indirect defence, which plants can obtain when they establish an obligate mutualism with ants.     

Potential sources of nitrogen in an ant-garden tank-bromeliad

Epiphytic plants in general and bromeliads in particular live in a water and nutrient-stressed environment often limited in nitrogen. Thus, these plants have developed different ways to survive in such an environment. We focused on Aechmea mertensii (Bromeliaceae), which is both a tank-bromeliad and an ant-garden (AG) epiphyte initiated by either the ants Camponotus femoratus or Pachycondyla goeldii. By combining a study of plant morphology and physiology associated with aquatic insect biology, we demonstrate that the ant species influences the leaf structure of the bromeliad, the structure of the aquatic community in its tank, and nutrient assimilation by the leaves. Based on nitrogen and nitrogen stable isotope measurements of the A. mertensii leaves, the leaf litter inside of the tank and the root-embedded carton nest, we discuss the potential sources of available nitrogen for the plant based on the ant partner. We demonstrate the existence of a complex ant-plant interaction that subsequently affects the biodiversity of a broader range of organisms that are themselves likely to influence nutrient assimilation by the A. mertensii leaves in a kind of plant-invertebrate-plant feedback loop.Key words: Aechmea mertensii, Camponotus femoratus, nitrogen, nitrogen stable isotope, Pachycondyla goeldii, plant-insect interactions, phytotelmataEpiphytes, which belong to numerous phylogenetically distant families, make up more than one-third of the total vascular plant biodiversity of neotropical rainforests.¹ Epiphytism implies physiological consequences and constraints resulting from the lack of access to what are by far the most important sources of water and nutrients for ground-rooting plants.² Epiphytic plants therefore have adapted in various ways to their aerial environment; adaptations for nutrient acquisition include growth forms such as “trash-baskets” (e.g., Asplenium), tanks (tank-bromeliads), specific leaf features such as absorbant trichomes (e.g., Tillandsia) and the velamen radicum in aerial roots (Orchidaceae).^1,3 Potential nitrogen sources for epiphytes may include (1) canopy-derived detritic nitrogen (mineralization of organic material from the canopy) and (2) atmospheric sources (wet and dry deposition, N₂ fixation),⁴ or may involve (3) interactions with animals.⁵ Indeed, some epiphytic species develop symbioses with ants, either by providing chambers (domatia) where ants nest or by rooting in ant gardens⁶ (AGs). By measuring differences in stable isotope composition, Treseder et al. (1995)⁷ estimated that the myrmecophyte Dischidia major (Asclepiadaceae) derives almost 30% of its nitrogen from debris deposited by the ants living in its domatia.Bromeliads are commonly found among the epiphytic species that root in ant-gardens. Their leaves form compartments acting as phytotelmata that hold rainwater and therefore provide habitats for aquatic macro- and microorganisms.^8,9 The detritus (e.g., windborne particulates, faeces, and dead leaves and animals) that enter the tanks constitute a source of nutrients for the aquatic food web, as well as for the bromeliad.¹⁰ Nitrogen is made available through the bacterial decomposition of organic matter, and the presence of arthropod predators in the phytotelmata food web most likely accelerates nitrogen cycling and leaf assimilation.²Recently, we described the complicated interaction that links an epiphytic ant-garden tank-bromeliad, its aquatic micro-ecosystem and the ants with which they are associated in the tropical forest of French Guiana.¹¹ Aechmea mertensii Schult.f. (Bromeliaceae) is an epiphytic tank-bromeliad that roots on AGs initiated either by the ants Camponotus femoratus Fabr. (living in a parabiotic association with Crematogaster levior Forel) or Pachychondyla goeldii Forel.¹² The AGs we studied were situated in tree canopies in pioneer growths along forest edges. By combining ecological studies characterizing the available light for the epiphyte, leaf morpho-anatomy, biochemical studies based on ¹⁵N isotope analysis and the richness of the aquatic communities inside of the tanks, we demonstrate the influence of the ant species on the foliar and aquatic community structures and, subsequently, on nitrogen acquisition by the plant.The ant species either directly or indirectly affects the light incidence, leaf structure, phytotelmata contents and nitrogen content of A. mertensii leaves, which is probably correlated to the plant''s fitness since nitrogen is a limiting factor for epiphytic plants. Pachycondyla goeldii and C. femoratus colonize exposed and partially-shaded areas, respectively. Exposed bromeliads (P. goeldii AGs) are smaller and limit direct light incidence by adopting an amphora-shape, whereas those growing in the shade (C. femoratus AGs) are larger and forage for light by developing a wider canopy. However, the contrast in the leaf mass area (LMA) response to light intensity based on the ant partner seems to support the hypothesis that leaf structure is not primarily controlled by light,^13,14 but rather directly by the ants or by other abiotic factors controlled by the ant partner. The availability of water and leaf debris from the surrounding vegetation may directly influence the diversity of the aquatic organisms in the phytotelmata that are themselves likely to influence nutrient assimilation by A. mertensii leaves in a kind of plant-invertebrate-plant feedback loop.Measurements of the percentage of total nitrogen showed that the carton nest, where the roots are embedded, was characterized by a greater amount of nitrogen compared to the leaf litter inside of the tank for both C. femoratus and P. goeldii AGs (Fig. 1A). While the nitrogen content remained constant for A. mertensii leaves and the leaf litter, the nitrogen content of the carton nest changed with the presence or absence of ants, and according to the ant partner. The carton nests of C. femoratus ants are richer in nitrogen than P. goeldii nests and non-occupied (i.e., abandoned) AGs, which have the lowest nitrogen content. Thus the sources of nitrogen could come from both the carton nest, where the roots are tightly embedded, and the phytotelmata with an average maximum nitrogen content of 2%. Delta ¹⁵N values enable us to estimate that A. mertensii may derive nitrogen from both ant species, but was affected differently depending on the ant partner (Fig. 1B). We demonstrate that A. mertensii—C. femoratus/Cr. levior associations can radically increase the amount of nutrients available to the host. Differences in stable N isotopic composition could reflect the diversity and richness of the macro- and microorganisms¹¹ in the phytotelm and/or the diversity of epiphytic species growing in the AGs,^12,15 and/or variations in the size of the ant colony living in the AGs. Indeed, C. femoratus/Cr. levior AGs are more densely populated than P. goeldii AGs.¹²Open in a separate windowFigure 1(A) Mean (±1 SE) of nitrogen content (%) and (B) δ¹⁵N (%) of Aechmea mertensii leaves (filled circles), root-embedded carton-nest (grey square) and phytotelmata leaf litter (empty triangle) for Camponotus femoratus (N = 10), Pachycondyla goeldii (N = 10) and non-occupied (N = 5) AGs.We have shown that AG ants provide nutrients to their host bromeliad. They either provide nitrogen-rich nutrients by directly or indirectly enhancing the nitrogen uptake of their host plants. Thus ants mediate nutrient uptake as well as phytotelmata contents. Furthermore, the strength of the mutualism appears to be dependent on the ant partner. In contrast to previous studies, our results showed that A. mertensii receives more than twice the amount of nitrogen compared to the myrmecophytic epiphyte Dischidia major⁷ or the epiphytic fern Antrophyum lanceolatum.¹⁶ Such a high level of insect-derived nitrogen could reflect the predominance of a dual nitrogen source derived from animals: one from the phytotelmata-associated organisms and one from the ant-debris (and faeces) in the carton nest. Nevertheless, further research on ¹⁵N enrichment is necessary to properly identify which part of the nitrogen input comes from animal remains and which part comes from the roots and phytotelmata. Moreover, it will enable us to identify which of the two AG ant species represents the greatest direct source of nitrogen for the plant.     

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.     

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     

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.     

Complete Genome Sequence of Croceibacter atlanticus HTCC2559T

Here we announce the complete genome sequence of Croceibacter atlanticus HTCC2559^T, which was isolated by high-throughput dilution-to-extinction culturing from the Bermuda Atlantic Time Series station in the Western Sargasso Sea. Strain HTCC2559^T contained genes for carotenoid biosynthesis, flavonoid biosynthesis, and several macromolecule-degrading enzymes. The genome confirmed physiological observations of cultivated Croceibacter atlanticus strain HTCC2559^T, which identified it as an obligate chemoheterotroph.The phylum Bacteroidetes comprises 6 to ∼30% of total bacterial communities in the ocean by fluorescence in situ hybridization (8-10). Most marine Bacteroidetes are in the family Flavobacteriaceae, most of which are aerobic respiratory heterotrophs that form a well-defined clade by 16S rRNA phylogenetic analyses (4). The members of this family are well known for degrading macromolecules, including chitin, DNA, cellulose, starch, and pectin (17), suggesting their environmental roles as detritus decomposers in the ocean (6). Marine Polaribacter and Dokdonia species in the Flavobacteriaceae have also shown to have photoheterotrophic metabolism mediated by proteorhodopsins (11, 12).Several strains of the family Flavobacteriaceae were isolated from the Sargasso Sea and Oregon coast, using high-throughput culturing approaches (7). Croceibacter atlanticus HTCC2559^T was cultivated from seawater collected at a depth of 250 m from the Sargasso Sea and was identified as a new genus in the family Flavobacteriaceae based on its 16S rRNA gene sequence similarities (6). Strain HTCC2559^T met the minimal standards for genera of the family Flavobacteriaceae (3) on the basis of phenotypic characteristics (6).Here we report the complete genome sequence of Croceibacter atlanticus HTCC2559^T. The genome sequencing was initiated by the J. Craig Venter Institute as a part of the Moore Foundation Microbial Genome Sequencing Project and completed in the current announcement. Gaps among contigs were closed by Genotech Co., Ltd. (Daejeon, Korea), using direct sequencing of combinatorial PCR products (16). The HTCC2559^T genome was analyzed with a genome annotation system based on GenDB (14) at Oregon State University and with the NCBI Prokaryotic Genomes Automatic Annotation Pipeline (15, 16).The HTCC2559^T genome is 2,952,962 bp long, with 33.9 mol% G+C content, and there was no evidence of plasmids. The number of protein-coding genes was 2,715; there were two copies of the 16S-23S-5S rRNA operon and 36 tRNA genes. The HTCC2559^T genome contained genes for a complete tricarboxylic acid cycle, glycolysis, and a pentose phosphate pathway. The genome also contained sets of genes for metabolic enzymes involved in carotenoid biosynthesis and also a serine/glycine hydroxymethyltransferase, which is often associated with the assimilatory serine cycle (13). The potential for HTCC2559^T to use bacterial type III polyketide synthase (PKS) needs to be confirmed because this organism had a naringenin-chalcone synthase (CHS) or chalcone synthase (EC 2.3.1.74), a key enzyme in flavonoid biosynthesis. CHS initiates the addition of three molecules of malonyl coenzyme A (malonyl-CoA) to a starter CoA ester (e.g., 4-coumaroyl-CoA) (1) and takes part in a few bacterial type III polyketide synthase systems (1, 2, 5, 18).The complete genome sequence confirmed that strain HTCC2559^T is an obligate chemoheterotroph because no genes for phototrophy were found. As expected from physiological characteristics (6), the HTCC2559^T genome contained a set of genes coding for enzymes required to degrade high-molecular-weight compounds, including peptidases, metallo-/serine proteases, pectinase, alginate lyases, and α-amylase.     

Colors of young and old spring leaves as a potential signal for ant-tended hemipterans

A new hypothesis explaining the adaptive significance of bright autumn leaf colors argues that these colors signal tree quality to myrmecophilous specialist aphids. In turn, the aphids attract aphid-tending ants during the following spring, which defend the trees from other aphids and herbivores. In this context, other types of plant coloration, such as the color change observed in young and old spring leaves, may function as a signal of plant quality for aphids and other myrmecophilous hemipterans. If these plant colors are costly for plants, then vividly colorful plants would be required to invest more in growth than in defense; as a result, colorful plants may be more palatable for honeydew-producing hemipterans, such as aphids, scale insects and treehoppers, although the relative importance of hemipterans other than aphids may be relatively low. These hemipterans may be attracted to colorful plants, after which their attendant ants would protect the plants from herbivory. However, it is necessary to examine color vision in hemipterans to support this hypothesis.Key words: ant-Hemiptera interactions, indirect effects, myrmecophiles, plant-ant mutualism, plant coloration, tritrophic interactionsRecently, the adaptive significance of plant coloration has attracted scientific interest.¹ Various theories have been postulated to explain the adaptive value of autumn leaf colors (red and yellow).² The coevolution hypothesis, the most novel and challenging theory among those proposed, argues that bright leaf colors serve as a conspicuous defense signal against autumn-colonizing insect herbivores, particularly aphids.³ According to this hypothesis, the production of autumn color pigments is an indicator of a particularly vigorous tree. Aphids, which have color vision and have long been associated with trees, migrate to winter host trees in the autumn and cause substantial damage. Therefore, vivid leaf color in the autumn would encourage aphids to colonize other less vigorously defended trees.⁴ Hamilton and Brown³ and Holopainen and Peltonen⁵ detected a higher number of specialist aphids on tree species with more intense autumn colors.After Hamilton and Brown,³ several researchers have attempted to explain the relationship between aphids and autumn color.^2,6 However, they did not account for several possibilities.⁶ First, healthy, vigorous trees may not be well defended, because they invest more in growth than in defense. Second, some aphid species avoid colonizing trees with bright colors, whereas others are attracted to bright colors. Finally, there are numerous multispecific interactions between plants, herbivores, predators and parasitoids in tree crowns. Ants prey on various arthropods living in trees, and ant-aphid mutualism affects arboreal arthropod communities. I incorporated these factors and formed a hypothesis in which autumn leaf colors signal tree quality to myrmecophilous specialist aphids. These aphids, in turn, attract aphid-tending ants during the following spring, which then defend the trees from other aphids and herbivores. Thus, autumn colors may be adaptive, because they attract myrmecophilous specialist aphids and their attendant ants, thereby reducing herbivory and interspecific competition among aphids.⁶In this addendum, I extend my former hypothesis beyond the relationship between autumn leaf colors and aphids. First, myrmecophilous aphids are not the only arthropods that benefit trees. Styrsky and Eubanks⁷ recently reviewed the literature regarding the effects of interactions between ants and honeydew-producing hemipterans on plants, and found that plants actually benefited indirectly from these interactions in most cases. This finding supports a new hypothesis focused on plant-ant mutualism via aphids. In addition, the mutualism between ants and honeydew-producing hemipterans includes many other organisms in addition to aphids, such as scale insects and treehoppers. Scale insects, especially soft scales (Coccidae) and mealybugs (Pseudococcidae), comprise many species that are tended by honeydew-collecting ants,⁸ and ant-scale insect mutualism is often beneficial for host plants.⁷ Although the female adults of scale insects are usually immobile, first-instar nymphs (crawlers) disperse by wind and locate on host plants, usually trees.⁹ The nymphs, emerging at various times from spring to autumn,¹⁰ may use plant coloration to select a suitable host. However, because specialist coccids and mealybugs represent a minority among the speciose scale insects,10 coevolutionary relationships between plants and ants via specialist scale insects may be relatively rare. The treehoppers also comprise many myrmecophilous species,^8,11 but the diversity of this group is highest in tropical regions; only a relatively small number of membracid species are present in temperate regions.¹² Therefore, scale insects and treehoppers may be attracted to autumn colors, and their attendant ants may then defend trees against other herbivorous insects. To fully account for the adaptive value of autumn colors, one would expect the importance of these hemipterans to be less than that of aphids, based on their low host-plant specificity, restricted distribution and life cycles. However, hemipterans may be associated with plant coloration in other aspects than autumn leaf color.Second, the colors of young and old spring leaves may also signal plant quality to ant-tended honeydew-producing hemipterans. The young leaves of many plants are reddish or yellowish (Fig. 1A and B).¹³ In the spring and other seasons, the old leaves of some evergreen tree species turn red or yellow (Fig. 1B). Because changes in leaf color may occur from spring to autumn, various hemipteran species may play specific roles as the season progresses. Aphids migrate in the spring and in the autumn,¹⁴ although most host-alternating aphids migrate to trees in autumn and to herbs in the late spring in temperate regions.¹⁵ If plants pay some cost for these colors¹⁶ and vivid colors indicate high plant quality for hemipterans, then changing colors may attract myrmecophilous hemipterans including aphids, scale insects and treehoppers, which may then protect plants against herbivory by other insects.Open in a separate windowFigure 1(A) Red young leaves of the evergreen oak Quercus glauca. (B) Yellowish young and reddish old leaves of the camphor tree Cinnamomum camphora.However, color vision has not been examined in detail in most hemipteran insects.^17,18 Many insects are insensitive to red, although one species of flower-visiting thrip is specifically attracted to red flowers.¹⁹ Thus, studies on color vision in hemipteran insects are required to evaluate this new hypothesis, as well as the coevolution hypothesis.     

Timing the switch to phototrophic growth: A possible role of GUN1

In young Arabidopsis seedlings, retrograde signaling from plastids regulates the expression of photosynthesis-associated nuclear genes in response to the developmental and functional state of the chloroplasts. The chloroplast-located PPR protein GUN1 is required for signalling following disruption of plastid protein synthesis early in seedling development before full photosynthetic competence has been achieved. Recently we showed that sucrose repression and the correct temporal expression of LHCB1, encoding a light-harvesting chlorophyll protein associated with photosystem II, are perturbed in gun1 mutant seedlings.¹ Additionally, we demonstrated that in gun1 seedlings anthocyanin accumulation and the expression of the “early” anthocyanin-biosynthesis genes is perturbed. Early seedling development, predominantly at the stage of hypocotyl elongation and cotyledon expansion, is also affected in gun1 seedlings in response to sucrose, ABA and disruption of plastid protein synthesis by lincomycin. These findings indicate a central role for GUN1 in plastid, sucrose and ABA signalling in early seedling development.Key words: ABA, ABI4, anthocyanin, chloroplast, GUN1, retrograde signalling, sucroseArabidopsis seedlings develop in response to light and other environmental cues. In young seedlings, development is fuelled by mobilization of lipid reserves until chloroplast biogenesis is complete and the seedlings can make the transition to phototrophic growth. The majority of proteins with functions related to photosynthesis are encoded by the nuclear genome, and their expression is coordinated with the expression of genes in the chloroplast genome. In developing seedlings, retrograde signaling from chloroplasts to the nucleus regulates the expression of these nuclear genes and is dependent on the developmental and functional status of the chloroplast. Two classes of gun (genomes uncoupled) mutants defective in retrograde signalling have been identified in Arabidopsis: the first, which comprises gun2–gun5, involves mutations in genes encoding components of tetrapyrrole biosynthesis.^2,3 The other comprises gun1, which has mutations in a nuclear gene encoding a plastid-located pentatricopeptide repeat (PPR) protein with an SMR (small MutS-related) domain near the C-terminus.^4,5 PPR proteins are known to have roles in RNA processing⁶ and the SMR domain of GUN1 has been shown to bind DNA,⁴ but the specific functions of these domains in GUN1 are not yet established. However, GUN1 has been shown to be involved in plastid gene expression-dependent,⁷ redox,⁴ ABA1,⁴ and sucrose signaling,^1,4,8 as well as light quality and intensity sensing pathways.^9–11 In addition, GUN1 has been shown to influence anthocyanin biosynthesis, hypocotyl extension and cotyledon expansion.^1,11     

Human non-CG methylation: Are human stem cells plant-like?

Non-CG methylation is well characterized in plants where it appears to play a role in gene silencing and genomic imprinting. Although strong evidence for the presence of non-CG methylation in mammals has been available for some time, both its origin and function remain elusive. In this review we discuss available evidence on non-CG methylation in mammals in light of evidence suggesting that the human stem cell methylome contains significant levels of methylation outside the CG site.Key words: non-CG methylation, stem cells, Dnmt1, Dnmt3a, human methylomeIn plant cells non-CG sites are methylated de novo by Chromomethylase 3, DRM1 and DRM2. Chromomethylase 3, along with DRM1 and DRM2 combine in the maintenance of methylation at symmetric CpHpG as well as asymmetric DNA sites where they appear to prevent reactivation of transposons.¹ DRM1 and DRM2 modify DNA de novo primarily at asymmetric CpH and CpHpH sequences targeted by siRNA.²Much less information is available on non-CG methylation in mammals. In fact, studies on mammalian non-CG methylation form a tiny fraction of those on CG methylation, even though data for cytosine methylation in other dinucleotides, CA, CT and CC, have been available since the late 1980s.³ Strong evidence for non-CG methylation was found by examining either exogenous DNA sequences, such as plasmid and viral integrants in mouse and human cell lines,^4,5 or transposons and repetitive sequences such as the human L1 retrotransposon⁶ in a human embryonic fibroblast cell line. In the latter study, non-CG methylation observed in L1 was found to be consistent with the capacity of Dnmt1 to methylate slippage intermediates de novo.⁶Non-CG methylation has also been reported at origins of replication^7,8 and a region of the human myogenic gene Myf3.⁹ The Myf3 gene is silenced in non-muscle cell lines but it is not methylated at CGs. Instead, it carries several methylated cytosines within the sequence CCTGG. Gene-specific non-CG methylation was also reported in a study of lymphoma and myeloma cell lines not expressing many B lineage-specific genes.¹⁰ The study focused on one specific gene, B29 and found heavy CG promoter methylation of that gene in most cell lines not expressing it. However, in two other cell lines where the gene was silenced, cytosine methylation was found almost exclusively at CCWGG sites. The authors provided evidence suggesting that CCWGG methylation was sufficient for silencing the B29 promoter and that methylated probes based on B29 sequences had unique gel shift patterns compared to non-methylated but otherwise identical sequences.¹⁰ The latter finding suggests that the presence of the non-CG methylation causes changes in the proteins able to bind the promoter, which could be mechanistically related to the silencing seen with this alternate methylation.Non-CG methylation is rarely seen in DNA isolated from cancer patients. However, the p16 promoter region was reported to contain both CG and non-CG methylation in breast tumor specimens but lacked methylation at these sites in normal breast tissue obtained at mammoplasty.¹¹ Moreover, CWG methylation at the CCWGG sites in the calcitonin gene is not found in normal or leukemic lymphocyte DNA obtained from patients.¹² Further, in DNA obtained from breast cancer patients, MspI sites that are refractory to digestion by MspI and thus candidates for CHG methylation were found to carry CpG methylation.¹³ Their resistance to MspI restriction was found to be caused by an unusual secondary structure in the DNA spanning the MspI site that prevents restriction.¹³ This latter observation suggests caution in interpreting EcoRII/BstNI or EcoRII/BstOI restriction differences as due to CWG methylation, since in contrast to the 37°C incubation temperature required for full EcoRII activity, BstNI and BstOI require incubation at 60°C for full activity where many secondary structures are unstable.The recent report by Lister et al.¹⁴ confirmed a much earlier report by Ramsahoye et al.¹⁵ suggesting that non-CG methylation is prevalent in mammalian stem cell lines. Nearest neighbor analysis was used to detect non-CG methylation in the earlier study on the mouse embryonic stem (ES) cell line,¹⁵ thus global methylation patterning was assessed. Lister et al.¹⁴ extend these findings to human stem cell lines at single-base resolution with whole-genome bisulfite sequencing. They report¹⁴ that the methylome of the human H1 stem cell line and the methylome of the induced pluripotent IMR90 (iPS) cell line are stippled with non-CG methylation while that of the human IMR90 fetal fibroblast cell line is not. While the results of the two studies are complementary, the human methylome study addresses locus specific non-CG methylation. Based on that data,¹⁴ one must conclude that non-CG methylation is not carefully maintained at a given site in the human H1 cell line. The average non-CG site is picked up as methylated in about 25% of the reads whereas the average CG methylation site is picked up in 92% of the reads. Moreover, non-CG methylation is not generally present on both strands and is concentrated in the body of actively transcribed genes.¹⁴Even so, the consistent finding that non-CG methylation appears to be confined to stem cell lines,^14,15 raises the possibility that cancer stem cells¹⁶ carry non-CG methylation while their nonstem progeny in the tumor carry only CG methylation. Given the expected paucity of cancer stem cells in a tumor cell population, it is unlikely that bisulfite sequencing would detect non-CG methylation in DNA isolated from tumor cells since the stem cell population is expected to be only a very minor component of tumor DNA. Published sequences obtained by bisulfite sequencing generally report only CG methylation, and to the best of our knowledge bisulfite sequenced tumor DNA specimens have not reported non-CG methylation. On the other hand, when sequences from cell lines have been reported, bisulfite-mediated genomic sequencing⁸ or ligation mediated PCR¹⁷ methylcytosine signals outside the CG site have been observed. In a more recent study plasmid DNAs carrying the Bcl2-major breakpoint cluster¹⁸ or human breast cancer DNA¹³ treated with bisulfite under non-denaturing conditions, cytosines outside the CG side were only partially converted on only one strand¹⁸ or at a symmetrical CWG site.¹³ In the breast cancer DNA study the apparent CWG methylation was not detected when the DNA was fully denatured before bisulfite treatment.¹³In both stem cell studies, non-CG methylation was attributed to the Dnmt3a,^14,15 a DNA methyltransferase with similarities to the plant DRM methyltransferase family¹⁹ and having the capacity to methylate non-CG sites when expressed in Drosophila melanogaster.¹⁵ DRM proteins however, possess a unique permuted domain structure found exclusively in plants¹⁹ and the associated RNA-directed non-CG DNA methylation has not been reproducibly observed in mammals despite considerable published^20–23 and unpublished efforts in that area. Moreover, reports where methylation was studied often infer methylation changes from 5AzaC reactivation studies²⁴ or find that CG methylation seen in plants but not non-CG methylation is detected.^21,22,25,26 In this regard, it is of interest that the level of non-CG methylation reported in stem cells corresponds to background non-CG methylation observed in vitro with human DNA methyltransferase I,²⁷ and is consistent with the recent report that cultured stem cells are epigenetically unstable.²⁸The function of non-CG methylation remains elusive. A role in gene expression has not been ruled out, as the studies above on Myf3 and B29 suggest.^9,10 However, transgene expression of the bacterial methyltransferase M.EcoRII in a human cell line (HK293), did not affect the CG methylation state at the APC and SerpinB5 genes²⁹ even though the promoters were symmetrically de novo methylated at ^mCWGs within each CCWGG sequence in each promoter. This demonstrated that CG and non-CG methylation are not mutually exclusive as had been suggested by earlier reports.^9,10 That observation is now extended to the human stem cell line methylome where CG and non-CG methylation co-exist.¹⁴ Gene expression at the APC locus was likewise unaffected by transgene expression of M.EcoRII. In those experiments genome wide methylation of the CCWGG site was detected by restriction analysis and bisulfite sequencing,²⁹ however stem cell characteristics were not studied.Many alternative functions can be envisioned for non-CG methylation, but the existing data now constrains them to functions that involve low levels of methylation that are primarily asymmetric. Moreover, inheritance of such methylation patterns requires low fidelity methylation. If methylation were maintained with high fidelity at particular CHG sites one would expect that the spontaneous deamination of 5-methylcytosine would diminish the number of such sites, so as to confine the remaining sites to those positions performing an essential function, as is seen in CG methylation.^30–33 However, depletion of CWG sites is not observed in the human genome.³⁴ Since CWG sites account for only about 50% of the non-CG methylation observed in the stem cell methylome¹⁴ where methylated non-CG sites carry only about 25% methylation, the probability of deamination would be about 13% of that for CWG sites that are subject to maintenance methylation in the germ line. Since mutational depletion of methylated cytosines has to have its primary effect on the germ line, if the maintenance of non-CG methylation were more accurate and more widespread, one would have had to argue that stem cells in the human germ lines lack CWG methylation. As it is the data suggests that whatever function non-CG methylation may have in stem cells, it does not involve accurate somatic inheritance in the germ line.The extensive detail on non-CG methylation in the H1 methylome¹⁴ raises interesting questions about the nature of this form of methylation in human cell lines. A key finding in this report is the contrast between the presence of non-CG methylation in the H1 stem cell line and its absence in the IMR90 human fetal lung fibroblast cell line.¹⁴ This suggests that it may have a role in the origin and maintenance of the pluripotent lineage.¹⁴By analogy with the well known methylated DNA binding proteins specific for CG methylation,³⁵ methylated DNA binding proteins that selectively bind sites of non-CG methylation are expected to exist in stem cells. Currently the only protein reported to have this binding specificity is human Dnmt1.^36–38 While Dnmt1 has been proposed to function stoichiometrically³⁹ and could serve a non-CG binding role in stem cells, this possibility and the possibility that other stem-cell specific non-CG binding proteins might exist remain to be been explored.Finally, the nature of the non-CG methylation patterns in human stem cell lines present potentially difficult technical problems in methylation analysis. First, based on the data in the H1 stem cell methylome,⁴⁰ a standard MS-qPCR for non-CG methylation would be impractical because non-CG sites are infrequent, rarely clustered and are generally characterized by partial asymmetric methylation. This means that a PCR primer that senses the 3 adjacent methylation sites usually recommended for MS-qPCR primer design^41,42 cannot be reliably found. For example in the region near Oct4 (Chr6:31,246,431), a potential MS-qPCR site exists with a suboptimal set of two adjacent CHG sites both methylated on the + strand at Chr6:31,252,225 and 31,252,237.^14,40 However these sites were methylated only in 13/45 and 30/52 reads. Thus the probability that they would both be methylated on the same strand is about 17%. Moreover, reverse primer locations containing non-CG methylation sites are generally too far away for practical bisulfite mediated PCR. Considering the losses associated with bisulfite mediated PCR⁴³ the likelihood that such an MS-qPCR system would detect non-CG methylation in the H1 cell line or stem cells present in a cancer stem cell niche^44,45 is very low.The second difficulty is that methods based on the specificity of MeCP2 and similar methylated DNA binding proteins for enriching methylated DNA (e.g., MIRA,⁴⁶ COMPARE-MS⁴⁷) will discard sequences containing non-CG methylation since they require cooperative binding afforded by runs of adjacent methylated CG sites for DNA capture. This latter property of the methylated cytosine capture techniques makes it also unlikely that methods based on 5-methylcytosine antibodies (e.g., meDIP⁴⁸) will capture non-CG methylation patterns accurately since the stem cell methylome shows that adjacent methylated non-CG sites are rare in comparison to methylated CG sites.¹⁴In summary, whether or not mammalian stem cells in general or human stem cells in particular possess functional plant-like methylation patterns is likely to continue to be an interesting and challenging question. At this point we can conclude that the non-CG patterns reported in human cells appear to differ significantly from the non-CG patterns seen in plants, suggesting that they do not have a common origin or function.     

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 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     

When do arbuscular mycorrhizal fungi protect plant roots from pathogens?

Arbuscular mycorrhizal (AM) fungi are mainly thought to facilitate phosphorus uptake in plants, but they can also perform several other functions that are equally beneficial. Our recent study sheds light on the factors determining one such function, enhanced plant protection from root pathogens. Root infection by the fungal pathogen Fusarium oxysporum was determined by both plant susceptibility and the ability of an AM fungal partner to suppress the pathogen. The non-susceptible plant species (Allium cepa) had limited F. oxysporum infection even without AM fungi. In contrast, the susceptible plant species (Setaria glauca) was heavily infected and only AM fungi in the family Glomeraceae limited pathogen abundance. Plant susceptibility to pathogens was likely determined by contrasting root architectures between plants, with the simple rooted plant (A. cepa) presenting fewer sites for infection. AM fungal colonization, however, was not limited in the same way in part because plants with fewer, simple roots are more mycorrhizal dependent. Protection only by Glomus species also indicates that whatever the mechanism(s) of this function, it responds to AM fungal families differently. While poor at pathogen protection, AM fungal species in the family Gigasporaceae most benefited the growth of the simple rooted plant species. Our research indicates that plant trait differences, such as root architecture can determine how important each mycorrhizal function is to plant growth but the ability to provide these functions differs among AM fungi.Key words: arbuscular mycorrhizal fungi, Fusarium oxysporum, root architecture, pathogen protection, multi-functionalityArbuscular mycorrhizas (AM) represent the oldest and most widespread symbiosis with land plants.¹ Most mycorrhizal research has focused on the ability of AM fungi to facilitate nutrient uptake, particularly phosphorus.² Although researchers recognize that AM fungi are multi-functional,³ it is not clear what factors determine which function an AM fungus performs or its relative importance to the plant.⁴ Newsham et al. (1995)³ hypothesized that AM function is based on root architecture: plants with simple rooting systems are dependent on mycorrhizas for nutrient uptake, while those with complex root systems are less dependent on mycorrhizas for nutrient uptake, but are more susceptible to root pathogens because of increased numbers of infections sites.³ These two functions, phosphorus uptake and enhanced pathogen protection from mycorrhizas also depend on the identity of the fungus. Arbuscular mycorrhizal fungi in the family Gigasporaceae are more effective at enhancing plant phosphorus, while AM fungi in the Glomeraceae better protect plants from root pathogens.⁵Our results support both plant and fungal control of a common pathogen, Fusarium oxysporum, and the interaction between these two factors ultimately determined the level of pathogen infection and plant mycorrhizal benefit. We inoculated two plant species that have contrasting root architectures with one of six AM fungal species from two families (or no AM fungi). After five months of growth, plants were inoculated with F. oxysporum, grown for another month and then harvested. All plant seeds and fungi were collected in a local old field community.⁶ Allium cepa (garden onion) was not susceptible to F. oxysporum likely because it has only a few adventitious roots below the main bulb that do not present many sites for infection. In contrast, Setaria glauca (yellow foxtail) was heavily infected by F. oxysporum and has fine roots with increased numbers of branching points and lateral meristems where fungi can colonize.⁷ For the susceptible plant (S. glauca), AM fungal species from the family Glomeraceae were effective at reducing pathogen abundance while species from the Gigasporaceae were not. Forming a symbiosis with a Glomus species resulted in S. glauca plants that were as large as control plants. AM fungal species from the family Gigaspoaceae were more beneficial to growth of the simple rooted A. cepa, which had fewer roots to take up soil nutrients.Reduced rooting structures may limit pathogen infection sites, but AM fungal colonization was not limited in the same way and may actually alter plant root architecture. While the simple rooted A. cepa had limited pathogen susceptibility, it had twice the AM fungal colonization of the complex rooted S. glauca. Because the simple rooted plant has a greater dependence on mycorrhizas,⁸ it likely transmits chemical signals to rapidly initiate mycorrhizal formation,⁹ but then may have less control on the spread of AM fungi within the root. In contrast, S. glauca is more susceptible to fungal pathogens and may be less mycorrhizal dependent in nature.¹⁰ As a result, S. glauca may treat all colonizing root fungi as potential parasites. Colonization by AM fungi from the Glomeraceae was also much greater than those in the Gigasporaceae due to differences in fungal life history strategy between these families.^11,12 AM fungal colonization can reduce root branching in plants and alter plant allocation to roots, thereby increasing mycorrhizal dependence for nutrients^10,13 and potentially reducing pathogen infection sites. Mycorrhizal induced changes to plant root architecture may therefore reinforce current mycorrhizal associations and alter future fungal colonization attempts.¹⁴ An important next step is to test if AM fungal families (or species) alter plant root architecture in different ways and the degree to which these effects depend on colonization timing and the plant host.Our study did not isolate the particular mechanism by which AM fungi control pathogens, but this mechanism clearly differentiates between AM fungal families. AM fungi can control pathogens through several mechanisms including direct competition for colonization sites, indirect initiation of plant defensive responses or altering other rhizosphere biota.¹⁵ Although these AM fungal families differ in the intensity of root colonization,¹¹ percentage of root length colonized by an AM fungus is a poor predictor of pathogen limitation compared to family identity,^12,16 suggesting that direct competition for space is unlikely. AM fungi share many cell surface molecules with pathogenic fungi like Fusarium.¹⁷ These molecules can act as signals that initiate plant production of defensive compounds such as phytoalexins, phenolics and other compounds.¹⁸ While AM fungi appear to evade these defenses, only AM fungal species in the family Glomeraceae would have elicited plant responses which altered future infection by F. oxysporum. AM fungi in the Gigasporaceae may differ more from F. oxysporum in their chemical signals or not colonize roots sufficiently to induce a sustained, system-wide plant response. In addition, many rhizosphere related microbes are antagonistic to pathogenic fungi¹⁵ and may differ in their response to the different AM fungal families.¹⁹ Because rhizosphere microbes also differ among plant species, plant pathogen protection may be influenced by multiple ecological interactions that determine the specific cases when mycorrhizal pathogen protection occurs. To distinguish between these mechanisms, future experiments could test whether biochemical similarity or ecological similarity (especially with other soil biota) between an AM fungus and fungal pathogen can predict mycorrhizal induced pathogen protection.Plant and fungal identity clearly affect AM fungal function and benefit, but to accurately use AM fungi in agriculture and restoration^20,21 we must clearly understand how functional mechanisms differ. Different mycorrhizal functions may be based on common plant traits like root architecture, but ecology, colonization timing and environment may alter the specific function AM fungi provide and its importance to plants. While it may be useful to establish greenhouse rules about which fungal species perform specific mycorrhizal functions, predicting their role in more complex systems relies on understanding if other factors will enhance or negate these effects. Most AM fungal species vary in their ability to perform each function and these can be locally adapted to limiting soil nutrients.²² In plants, there is also a range to which specific mycorrhizal functions may benefit plant fitness, and these responses are based on both plant traits (which change throughout a plant''s life cycle) and the local environment.^23,24 Given this variation, it is critical to understand if AM fungi can respond to cues from the plant or the environment to identify what factors limit plant growth and whether a the most effective AM fungus shows a greater response.