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Background:
In previous studies, the gram-positive firmicute genus Paenibacillus was found with significant abundances in nests of wild solitary bees. Paenibacillus larvae is well-known for beekeepers as a severe pathogen causing the fatal honey bee disease American foulbrood, and other members of the genus are either secondary invaders of European foulbrood or considered a threat to honey bees. We thus investigated whether Paenibacillus is a common bacterium associated with various wild bees and hence poses a latent threat to honey bees visiting the same flowers.
Results:
We collected 202 samples from 82 individuals or nests of 13 bee species at the same location and screened each for Paenibacillus using high-throughput sequencing-based 16S metabarcoding. We then isolated the identified strain Paenibacillus MBD-MB06 from a solitary bee nest and sequenced its genome. We did find conserved toxin genes and such encoding for chitin-binding proteins, yet none specifically related to foulbrood virulence or chitinases. Phylogenomic analysis revealed a closer relationship to strains of root-associated Paenibacillus rather than strains causing foulbrood or other accompanying diseases. We found anti-microbial evidence within the genome, confirmed by experimental bioassays with strong growth inhibition of selected fungi as well as gram-positive and gram-negative bacteria.
Conclusions:
The isolated wild bee associate Paenibacillus MBD-MB06 is a common, but irregularly occurring part of wild bee microbiomes, present on adult body surfaces and guts and within nests especially in megachilids. It was phylogenetically and functionally distinct from harmful members causing honey bee colony diseases, although it shared few conserved proteins putatively toxic to insects that might indicate ancestral predisposition for the evolution of insect pathogens within the group. By contrast, our strain showed anti-microbial capabilities and the genome further indicates abilities for chitin-binding and biofilm-forming, suggesting it is likely a useful associate to avoid fungal penetration of the bee cuticula and a beneficial inhabitant of nests to repress fungal threats in humid and nutrient-rich environments of wild bee nests.
Summary Using the facultative root hemiparasite Rhinanthus minor and Hordeum vulgare as a host, several aspects of water relations, the flows and partitioning of mineral nutrients, the flows, depositions and metabolism of abscisic acid (ABA) and zeatin type cytokinins (zeatin Z, zeatin riboside ZR, zeatin nucleotide ZN) within the host, the parasite and between host and parasite and the flows and partitioning of the transport metabolites mannitol in the parasite, and of sucrose in the host, have been studied during the study period 41 to 54 days after planting, i.e about 30 to 43 days after successful attachment of the parasite to the host. Water relations Extraction of xylem sap by the parasite from the host’s roots is facilitated by considerably higher transpiration per leaf area in the parasite than in the host and by the fact that stomata of attached Rhinanthus were wide open all day and night despite extremely high ABA concentrations in the leaves. By comparison, another related root hemiparasite, Melampyrum arvense, parasitising on various grasses in the field (botanic garden), showed normal diurnal stomatal behaviour. The abnormal behaviour of Rhinanthus stomata was not due to anatomical reasons as closure could be induced by applying high external ABA concentrations. Remarkable differences have been detected between the hydraulic conductance of barley seminal roots showing relatively low values, and that of Rhinanthus the seminal root showing very high values. The latter could be related to the observed high ABA concentrations in these roots. Whole plant water uptake, transpirational losses, growth-dependent deposition and the flows of water within the plants have been measured in singly growing Rhinanthus and Hordeum plants and in the parasitic association between the two. Water uptake, deposition and transpiration in Rhinanthus were dramatically increased after attachment to the barley host; most of the water used by the parasite was extracted as xylem sap from the host, thereby scavenging 20% of the total water taken up by the host’s roots. This water uptake by the parasitised host, however, due to a parasite induced reduction in the hosts growth, was decreased by 22% as compared to non- parasitised barley. The overall changes in growth-related water deposition in host and parasite pointed to decreased shoot and relatively favoured root growth in the host and to strongly favoured shoot growth and less strongly increased root growth only in the parasite. These changes in the host became more severe, when more than one Rhinanthus was parasitising one barley plant. Mineral nutrients relations 5 mM NO3- supply In parasitising Rhinanthus shoot growth was 12-fold, but root growth only twofold increased compared to the non-parasitising (very small) plants. On the other hand, in the Hordeum host, shoot dry matter growth was clearly reduced, by 33% in leaf laminae and by 52% in leaf sheaths, whereas root growth was only slightly reduced as a consequence of parasitism. Growth-dependent increments of total N and P and of K, Ca and Mg in parasitising Rhinanthus shoot were strongly increased, particularly increments of total N and P, which were 18 and 42 times, respectively, higher than in the small solitary Rhinanthus. On the other hand, increments of the above mineral nutrients in leaf sheaths of parasitised Hordeum vulgare were more strongly decreased than in leaf laminae in response to parasitic attack. Estimation of the flows of nutrients revealed that Rhinanthus withdrew from the host xylem sap about the same percentage of each nutrients: 18% of total N, 22% of P and 20% of K. Within the host almost all net flows of nutrient ions were decreased due to parasitism, but retranslocation from shoot to root-as related to xylem flow-was somewhat increased for all nutrients. Quantitative information is provided to show that the substantially increased growth in the shoot of attached Rhinanthus and the observed decrease in Hordeum shoot growth after infection were related to strongly elevated supply of nitrogen and phosphorus in the parasite and to incipient deficiency of these nutrients in the parasitised host. The flows of nutrients between host and parasite are discussed in terms of low selectivity of nutrient abstraction from the host xylem by the hemiparasite Rhinanthus minor. 1 mM NO3- or 1 mM NH4+ supply Rhinanthus shoot growth as measured by dry matter increase, was 19-fold (1 mM NO3-) and 15-fold (1 mM NH4+), but root growth only twofold (1 mM NO3-) and 2.9-fold (1 mM NH4+) increased-relative to singly growing Rhinanthus-when parasitising on host barley. In the Hordeum host, shoot dry matter growth was clearly reduced, whereas root growth was only slightly affected. Growth-dependent increments of total N and P and of K, Ca and Mg in parasitising Rhinanthus shoot were strongly increased, particularly increments of total N or of P, which were 20 or 53 times (1 mM NO3-) and 18 or 51 times (1 mM NH4+) , respectively, higher than those in solitary Rhinanthus. Within the host almost all net flows of nutrient ions were decreased due to parasitism. Flows of mannitol in parasite and sucrose flows in host barley When the plants were supplied with 5 mM NO3-, the biosynthesis of mannitol in Rhinanthus shoots increased 16-fold by parasitism, resulting in a 15-fold higher mannitol flow in the phloem and a 10-fold higher deposition in the shoot. Also the backward transport of mannitol in the xylem were increased 10-fold after attachment. Lower level nitrogen supply increased the deposition of mannitol in both single and attached Rhinanthus shoot and root. No mannitol was found in barley roots even in the direct vicinity of the haustoria. This indicates there are no backward transport of xylem sap from parasite to host. Compared to unparasitised barley, the net biosynthesis and deposition of sucrose in the shoot and the phloem flow was decreased substantially when plants were supplied with 5 mM NO3- or 1 mM NO3-. No sucrose has been detected in barley xylem sap and consequently there was no indication of a sucrose transfer from the host to the parasite. A possible involvement of mannitol in the abscisic acid relations of the parasite is discussed. ABA relations When the plants were supplied with 5 mM NO3-, there were weak or no effects of parasitism on ABA flows, biosynthesis and ABA degradation in barley. However, ABA growth-dependent deposition was significantly increased in the leaf laminae (3 fold) and in leaf sheath (2.4 fold), but not in roots. Dramatic changes in ABA flows, metabolism and deposition on a per plant basis, however, have been observed in Rhinanthus. Biosynthesis in the roots was 12-fold higher after attachment resulting in 14-fold higher ABA flows in the xylem. A large portion of this ABA was metabolised, a small portion was deposited. Phloem flows of ABA were increased 13-fold after attachment. The concentrations of ABA in tissues and xylem sap were higher in attached Rhinanthus by an order of magnitude than in host tissues and xylem sap. Similar dramatic difference existed when comparing the high concentrations in the xylem sap of single Rhinanthus with unparasitised barley. As compared to 5 mM NO3-, lower NO3- or 1 mM NH4+ supply doubled the ABA concentrations in barley leaf laminae, while having only small or no significant effects in the other organs. The possible special functions of ABA for the parasite are discussed. Zeatin type cytokinins relations Parasitism decreased, in the case of zeatin (Z), the synthesis (by 57%) in the root, xylem flows (by 56%) and metabolism (by 71%) in leaf laminae, however, increased the phloem flows of zeatin massively (3-fold) in host barley. The deposition of zeatin in the root of Rhinanthus and the flowing in xylem and phloem were 24, 12, 29-fold, respectively, increased after successfully attaching to the host barley. However, net biosynthesis of zeatin in Rhinanthus roots decreased by 39% after attachment. This indicates that a large portion (70%) of xylem flow of zeatin in attached Rhinanthus was extracted from the host. In singly growing Rhinanthus plants, the balance of zeatin deposition in the shoot was negative, i.e. zeatin was metabolised and exported back to root in the phloem. The xylem flows of zeatin riboside (ZR) in barley decreased by 39% after infected by Rhinanthus; phloem flow, which was 117% relative to xylem flow was less decreased (by 13%) after infection. Deposition of ZR has not been significantly affected in the leaf laminae, in leaf sheaths and roots. After parasitising on the host barley depositions in root, xylem flow and phloem flow increased 12, 18, 88–fold respectively in Rhinanthus. A large portion (57%) of xylem flow of ZR in attached Rhinanthus was extracted from the host. In single Rhinanthus increament of shoot zeatin riboside was negative and a substantial portion was degraded in shoot and the rest was retranslocated back to the root in the phloem. A significant depositions of Z and ZR were detected in the haustoria of the Rhinanthus/barley association. Flows and deposition of zeatin nucleotides also have been investigated. The possible physiological functions of the large quantities of Z and ZR derived from the host barley, for the improved growth and the stomatal opening in the parasitising Rhinanthus are discussed.
Farmland tree cultivation is considered an important option for enhancing wood production. In South India, the native leaf-deciduous tree species Melia dubia is popular for short-rotation plantations. Across a rainfall gradient from 420 to 2170 mm year\(^{–1}\), we studied 186 farmland woodlots between one and nine years in age. The objectives were to identify the main factors controlling aboveground biomass (AGB) and growth rates. A power-law growth model predicts an average stand-level AGB of 93.8 Mg ha\(^{–1}\) for nine-year-old woodlots. The resulting average annual AGB increment over the length of the rotation cycle is 10.4 Mg ha\(^{–1}\) year\(^{–1}\), which falls within the range reported for other tropical tree plantations. When expressing the parameters of the growth model as functions of management, climate and soil variables, it explains 65% of the variance in AGB. The results indicate that water availability is the main driver of the growth of M. dubia. Compared to the effects of water availability, the effects of soil nutrients are 26% to 60% smaller. We conclude that because of its high biomass accumulation rates in farm forestry, M. dubia is a promising candidate for short-rotation plantations in South India and beyond.
In contrast to the plasma membrane, the vacuole membrane has not yet been associated with electrical excitation of plants. Here, we show that mesophyll vacuoles from Arabidopsis sense and control the membrane potential essentially via the K\(^+\)-permeable TPC1 and TPK channels. Electrical stimuli elicit transient depolarization of the vacuole membrane that can last for seconds. Electrical excitability is suppressed by increased vacuolar Ca\(^{2+}\) levels. In comparison to wild type, vacuoles from the fou2 mutant, harboring TPC1 channels insensitive to luminal Ca\(^{2+}\), can be excited fully by even weak electrical stimuli. The TPC1-loss-of-function mutant tpc1-2 does not respond to electrical stimulation at all, and the loss of TPK1/TPK3-mediated K\(^{+}\) transport affects the duration of TPC1-dependent membrane depolarization. In combination with mathematical modeling, these results show that the vacuolar K\(^+\)-conducting TPC1 and TPK1/TPK3 channels act in concert to provide for Ca\(^{2+}\)- and voltage-induced electrical excitability to the central organelle of plant cells.
To fire action-potential-like electrical signals, the vacuole membrane requires the two-pore channel TPC1, formerly called SV channel. The TPC1/SV channel functions as a depolarization-stimulated, non-selective cation channel that is inhibited by luminal Ca\(^{2+}\). In our search for species-dependent functional TPC1 channel variants with different luminal Ca\(^{2+}\) sensitivity, we found in total three acidic residues present in Ca\(^{2+}\) sensor sites 2 and 3 of the Ca\(^{2+}\)-sensitive AtTPC1 channel from Arabidopsis thaliana that were neutral in its Vicia faba ortholog and also in those of many other Fabaceae. When expressed in the Arabidopsis AtTPC1-loss-of-function background, wild-type VfTPC1 was hypersensitive to vacuole depolarization and only weakly sensitive to blocking luminal Ca\(^{2+}\). When AtTPC1 was mutated for these VfTPC1-homologous polymorphic residues, two neutral substitutions in Ca\(^{2+}\) sensor site 3 alone were already sufficient for the Arabidopsis At-VfTPC1 channel mutant to gain VfTPC1-like voltage and luminal Ca\(^{2+}\) sensitivity that together rendered vacuoles hyperexcitable. Thus, natural TPC1 channel variants exist in plant families which may fine-tune vacuole excitability and adapt it to environmental settings of the particular ecological niche.
The animal diet of the carnivorous Venus flytrap, Dionaea muscipula, contains a sodium load that enters the capture organ via an HKT1-type sodium channel, expressed in special epithelia cells on the inner trap lobe surface. DmHKT1 expression and sodium uptake activity is induced upon prey contact. Here, we analyzed the HKT1 properties required for prey sodium osmolyte management of carnivorous Dionaea. Analyses were based on homology modeling, generation of model-derived point mutants, and their functional testing in Xenopus oocytes. We showed that the wild-type HKT1 and its Na\(^+\)- and K\(^+\)-permeable mutants function as ion channels rather than K\(^+\) transporters driven by proton or sodium gradients. These structural and biophysical features of a high-capacity, Na\(^+\)-selective ion channel enable Dionaea glands to manage prey-derived sodium loads without confounding the action potential-based information management of the flytrap.
Almost all life forms on earth have adapted to the most impactful and most predictable recurring change in environmental condition, the cycle of day and night, caused by the axial rotation of the planet. As a result many animals have evolved intricate endogenous clocks, which adapt and synchronize the organisms’ physiology, metabolism and behaviour to the daily change in environmental conditions. The scientific field researching these endogenous clocks is called chronobiology and has steadily grown in size, scope and relevance since the works of the earliest pioneers in the 1960s.
The number one model organism for the research of circadian clocks is the fruit fly, Drosophila melanogaster, whose clock serves as the entry point to understanding the basic inner workings of such an intricately constructed endogenous timekeeping system. In this thesis it was attempted to combine the research on the circadian clock with the techniques of optogenetics, a fairly new scientific field, launched by the discovery of Channelrhodopsin 2 just over 15 years ago. Channelrhodopsin 2 is a light-gated ion channel found in the green alga Chlamydomonas reinhardtii. In optogenetics, researches use these light-gated ion channels like Channelrhodopsin 2 by heterologously expressing them in cells and tissues of other organisms, which can then be stimulated by the application of light. This is most useful when studying neurons, as these channels provide an almost non-invasive tool to depolarize the neuronal plasma membranes at will. The goal of this thesis was to develop an optogenetic tool, which would be able to influence and phase shift the circadian clock of Drosophila melanogaster upon illumination. A phase shift is the adaptive response of the circadian clock to an outside stimulus that signals a change in the environmental light cycle. An optogenetic tool, able to influence and phase shift the circadian clock predictably and reliably, would open up many new ways and methods of researching the neuronal network of the clock and which neurons communicate to what extent, ultimately synchronizing the network.
The first optogenetic tool to be tested in the circadian clock of Drosophila melanogaster was ChR2-XXL, a channelrhodopsin variant with dramatically increased expression levels and photocurrents combined with a prolonged open state. The specific expression of ChR2-XXL and of later constructs was facilitated by deploying the three different clock-specific GAL4-driver lines, clk856-gal4, pdf-gal4 and mai179-gal4. Although ChR2-XXL was shown to be highly effective at depolarizing neurons, these stimulations proved to be unable to significantly phase shift the circadian clock of Drosophila. The second series of experiments was conducted with the conceptually novel optogenetic tools Olf-bPAC and SthK-bPAC, which respectively combine a cyclic nucleotide-gated ion channel (Olf and SthK) with the light-activated adenylyl-cyclase bPAC. These tools proved to be quite useful when expressed in the motor neurons of instar-3 larvae of Drosophila, paralyzing the larvae upon illumination, as well as affecting body length. This way, these new tools could be precisely characterized, spawning a successfully published research paper, centered around their electrophysiological characterization and their applicability in model organisms like Drosophila. In the circadian clock however, these tools caused substantial damage, producing severe arrhythmicity and anomalies in neuronal development. Using a temperature-sensitive GAL80-line to delay the expression until after the flies had eclosed, yielded no positive results either. The last series of experiments saw the use of another new series of optogenetic tools, modelled after the Olf-bPAC, with bPAC swapped out for CyclOp, a membrane-bound guanylyl-cyclase, coupled with less potent versions of the Olf. This final attempt however also ended up being unsuccessful. While these tools could efficiently depolarize neuronal membranes upon illumination, they were ultimately unable to stimulate the circadian clock in way that would cause it to phase shift.
Taken together, these mostly negative results indicate that an optogenetic manipulation of the circadian clock of Drosophila melanogaster is an extremely challenging subject. As light already constitutes the most impactful environmental factor on the circadian clock, the combination of chronobiology with optogenetics demands the parameters of the conducted experiments to be tuned with an extremely high degree of precision, if one hopes to receive positive results from these types of experiments at all.
Using Expansion Microscopy to Visualize and Characterize the Morphology of Mitochondrial Cristae
(2020)
Mitochondria are double membrane bound organelles indispensable for biological processes such as apoptosis, cell signaling, and the production of many important metabolites, which includes ATP that is generated during the process known as oxidative phosphorylation (OXPHOS). The inner membrane contains folds called cristae, which increase the membrane surface and thus the amount of membrane-bound proteins necessary for the OXPHOS. These folds have been of great interest not only because of their importance for energy conversion, but also because changes in morphology have been linked to a broad range of diseases from cancer, diabetes, neurodegenerative diseases, to aging and infection. With a distance between opposing cristae membranes often below 100 nm, conventional fluorescence imaging cannot provide a resolution sufficient for resolving these structures. For this reason, various highly specialized super-resolution methods including dSTORM, PALM, STED, and SIM have been applied for cristae visualization. Expansion Microscopy (ExM) offers the possibility to perform super-resolution microscopy on conventional confocal microscopes by embedding the sample into a swellable hydrogel that is isotropically expanded by a factor of 4–4.5, improving the resolution to 60–70 nm on conventional confocal microscopes, which can be further increased to ∼ 30 nm laterally using SIM. Here, we demonstrate that the expression of the mitochondrial creatine kinase MtCK linked to marker protein GFP (MtCK-GFP), which localizes to the space between the outer and the inner mitochondrial membrane, can be used as a cristae marker. Applying ExM on mitochondria labeled with this construct enables visualization of morphological changes of cristae and localization studies of mitochondrial proteins relative to cristae without the need for specialized setups. For the first time we present the combination of specific mitochondrial intermembrane space labeling and ExM as a tool for studying internal structure of mitochondria.
Soil salinity is an increasingly global problem which hampers plant growth and crop yield. Plant productivity depends on optimal water-use efficiency and photosynthetic capacity balanced by stomatal conductance. Whether and how stomatal behavior contributes to salt sensitivity or tolerance is currently unknown. This work identifies guard cell-specific signaling networks exerted by a salt-sensitive and salt-tolerant plant under ionic and osmotic stress conditions accompanied by increasing NaCl loads.
We challenged soil-grown Arabidopsis thaliana and Thellungiella salsuginea plants with short- and long-term salinity stress and monitored genome-wide gene expression and signals of guard cells that determine their function.
Arabidopsis plants suffered from both salt regimes and showed reduced stomatal conductance while Thellungiella displayed no obvious stress symptoms. The salt-dependent gene expression changes of guard cells supported the ability of the halophyte to maintain high potassium to sodium ratios and to attenuate the abscisic acid (ABA) signaling pathway which the glycophyte kept activated despite fading ABA concentrations.
Our study shows that salinity stress and even the different tolerances are manifested on a single cell level. Halophytic guard cells are less sensitive than glycophytic guard cells, providing opportunities to manipulate stomatal behavior and improve plant productivity.
Two-component cyclase opsins of green algae are ATP-dependent and light-inhibited guanylyl cyclases
(2018)
Background:
The green algae Chlamydomonas reinhardtii and Volvox carteri are important models for studying light perception and response, expressing many different photoreceptors. More than 10 opsins were reported in C. reinhardtii, yet only two—the channelrhodopsins—were functionally characterized. Characterization of new opsins would help to understand the green algae photobiology and to develop new tools for optogenetics.
Results:
Here we report the characterization of a novel opsin family from these green algae: light-inhibited guanylyl cyclases regulated through a two-component-like phosphoryl transfer, called “two-component cyclase opsins” (2c-Cyclops). We prove the existence of such opsins in C. reinhardtii and V. carteri and show that they have cytosolic N- and C-termini, implying an eight-transmembrane helix structure. We also demonstrate that cGMP production is both light-inhibited and ATP-dependent. The cyclase activity of Cr2c-Cyclop1 is kept functional by the ongoing phosphorylation and phosphoryl transfer from the histidine kinase to the response regulator in the dark, proven by mutagenesis. Absorption of a photon inhibits the cyclase activity, most likely by inhibiting the phosphoryl transfer. Overexpression of Vc2c-Cyclop1 protein in V. carteri leads to significantly increased cGMP levels, demonstrating guanylyl cyclase activity of Vc2c-Cyclop1 in vivo. Live cell imaging of YFP-tagged Vc2c-Cyclop1 in V. carteri revealed a development-dependent, layer-like structure at the immediate periphery of the nucleus and intense spots in the cell periphery.
Conclusions:
Cr2c-Cyclop1 and Vc2c-Cyclop1 are light-inhibited and ATP-dependent guanylyl cyclases with an unusual eight-transmembrane helix structure of the type I opsin domain which we propose to classify as type Ib, in contrast to the 7 TM type Ia opsins. Overexpression of Vc2c-Cyclop1 protein in V. carteri led to a significant increase of cGMP, demonstrating enzyme functionality in the organism of origin. Fluorescent live cell imaging revealed that Vc2c-Cyclop1 is located in the periphery of the nucleus and in confined areas at the cell periphery.