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- sphingolipid (1)
- sponge holicolona-simulans (1)
- sponge-associated actinomyetes (1)
- staphilococci (1)
- staurosporine (1)
- stress (1)
- stress signaling cascade (1)
- stress tolerance (1)
- striga (1)
- structured illumination microscope (1)
- subcutaneous animal model (1)
- sucrose (1)
- sugar absorption (1)
- sugar beet (1)
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- sugar transport protein (1)
- sulforaphane (1)
- superfamily (1)
- support vector machine (1)
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- symbiotic bacteria (1)
- synechocystis (1)
- synthetic D-glucose analogy (1)
- tannins (1)
- task allocation (1)
- taxonomy (1)
- temperature (1)
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- tomato (1)
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- trypanosoma brucei (1)
- tryptophan (1)
- tumor cell (1)
- two-component system (1)
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- type 2 diabetes (1)
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- unfolded protein response (1)
- vacuolar calcium sensor (1)
- vacuolar membrane (1)
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- volvox carteri (1)
- von Willebrand type C domain (1)
- water availability (1)
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- weed dodder (1)
- whole genome sequencing (1)
- whole-genome duplication (1)
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- xenobiotic metabolism (1)
- xylem loading (1)
Institute
- Julius-von-Sachs-Institut für Biowissenschaften (157) (remove)
Sonstige beteiligte Institutionen
Small bacterial regulatory RNAs (sRNAs) have been implicated in the regulation of numerous metabolic pathways. In most of these studies, sRNA-dependent regulation of mRNAs or proteins of enzymes in metabolic pathways has been predicted to affect the metabolism of these bacteria. However, only in a very few cases has the role in metabolism been demonstrated. Here, we performed a combined transcriptome and metabolome analysis to define the regulon of the sibling sRNAs NgncR_162 and NgncR_163 (NgncR_162/163) and their impact on the metabolism of Neisseria gonorrhoeae. These sRNAs have been reported to control genes of the citric acid and methylcitric acid cycles by posttranscriptional negative regulation. By transcriptome analysis, we now expand the NgncR_162/163 regulon by several new members and provide evidence that the sibling sRNAs act as both negative and positive regulators of target gene expression. Newly identified NgncR_162/163 targets are mostly involved in transport processes, especially in the uptake of glycine, phenylalanine, and branched-chain amino acids. NgncR_162/163 also play key roles in the control of serine-glycine metabolism and, hence, probably affect biosyntheses of nucleotides, vitamins, and other amino acids via the supply of one-carbon (C\(_1\)) units. Indeed, these roles were confirmed by metabolomics and metabolic flux analysis, which revealed a bipartite metabolic network with glucose degradation for the supply of anabolic pathways and the usage of amino acids via the citric acid cycle for energy metabolism. Thus, by combined deep RNA sequencing (RNA-seq) and metabolomics, we significantly extended the regulon of NgncR_162/163 and demonstrated the role of NgncR_162/163 in the regulation of central metabolic pathways of the gonococcus.
Honeybees (Apis mellifera) need their fine sense of taste to evaluate nectar and pollen sources. Gustatory receptors (Grs) translate taste signals into electrical responses. In vivo experiments have demonstrated collective responses of the whole Gr-set. We here disentangle the contributions of all three honeybee sugar receptors (AmGr1-3), combining CRISPR/Cas9 mediated genetic knock-out, electrophysiology and behaviour. We show an expanded sugar spectrum of the AmGr1 receptor. Mutants lacking AmGr1 have a reduced response to sucrose and glucose but not to fructose. AmGr2 solely acts as co-receptor of AmGr1 but not of AmGr3, as we show by electrophysiology and using bimolecular fluorescence complementation. Our results show for the first time that AmGr2 is indeed a functional receptor on its own. Intriguingly, AmGr2 mutants still display a wildtype-like sugar taste. AmGr3 is a specific fructose receptor and is not modulated by a co-receptor. Eliminating AmGr3 while preserving AmGr1 and AmGr2 abolishes the perception of fructose but not of sucrose. Our comprehensive study on the functions of AmGr1, AmGr2 and AmGr3 in honeybees is the first to combine investigations on sugar perception at the receptor level and simultaneously in vivo. We show that honeybees rely on two gustatory receptors to sense all relevant sugars.
Plants extract mineral nutrients from the soil, or from interactions with mutualistic soil microbes via their root systems. Adapting root architecture to nutrient availability enables efficient resource utilization, particularly in patchy and dynamic environments. Root growth responses to soil nitrogen levels are shoot-mediated, but the identity of shoot-derived mobile signals regulating root growth responses has remained enigmatic. Here we show that a shoot-derived micro RNA, miR2111, systemically steers lateral root initiation and nitrogen responsiveness through its root target TML (TOO MUCH LOVE) in the legume Lotus japonicus, where miR2111 and TML were previously shown to regulate symbiotic infections with nitrogen fixing bacteria. Intriguingly, systemic control of lateral root initiation by miR2111 and TML/HOLT (HOMOLOGUE OF LEGUME TML) was conserved in the nonsymbiotic ruderal Arabidopsis thaliana, which follows a distinct ecological strategy. Thus, the miR2111-TML/HOLT regulon emerges as an essential, conserved factor in adaptive shoot control of root architecture in dicots.
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.
Interleukin-4 (IL-4) plays a key role in atopic diseases. It coordinates T-helper cell differentiation to subtype 2, thereby directing defense toward humoral immunity. Together with Interleukin-13, IL-4 further induces immunoglobulin class switch to IgE. Antibodies of this type activate mast cells and basophilic and eosinophilic granulocytes, which release pro-inflammatory mediators accounting for the typical symptoms of atopic diseases. IL-4 and IL-13 are thus major targets for pharmaceutical intervention strategies to treat atopic diseases. Besides neutralizing antibodies against IL-4, IL-13, or its receptors, IL-4 antagonists can present valuable alternatives. Pitrakinra, an Escherichia coli-derived IL-4 antagonist, has been evaluated in clinical trials for asthma treatment in the past; however, deficits such as short serum lifetime and potential immunogenicity among others stopped further development. To overcome such deficits, PEGylation of therapeutically important proteins has been used to increase the lifetime and proteolytic stability. As an alternative, glycoengineering is an emerging strategy used to improve pharmacokinetics of protein therapeutics. In this study, we have established different strategies to attach glycan moieties to defined positions in IL-4. Different chemical attachment strategies employing thiol chemistry were used to attach a glucose molecule at amino acid position 121, thereby converting IL-4 into a highly effective antagonist. To enhance the proteolytic stability of this IL-4 antagonist, additional glycan structures were introduced by glycoengineering utilizing eucaryotic expression. IL-4 antagonists with a combination of chemical and biosynthetic glycoengineering could be useful as therapeutic alternatives to IL-4 neutralizing antibodies already used to treat atopic diseases.
Young grapevines (Vitis vinifera) suffer and eventually can die from the crown gall disease caused by the plant pathogen Allorhizobium vitis (Rhizobiaceae). Virulent members of A. vitis harbor a tumor-inducing plasmid and induce formation of crown galls due to the oncogenes encoded on the transfer DNA. The expression of oncogenes in transformed host cells induces unregulated cell proliferation and metabolic and physiological changes. The crown gall produces opines uncommon to plants, which provide an important nutrient source for A. vitis harboring opine catabolism enzymes. Crown galls host a distinct bacterial community, and the mechanisms establishing a crown gall–specific bacterial community are currently unknown. Thus, we were interested in whether genes homologous to those of the tumor-inducing plasmid coexist in the genomes of the microbial species coexisting in crown galls. We isolated 8 bacterial strains from grapevine crown galls, sequenced their genomes, and tested their virulence and opine utilization ability in bioassays. In addition, the 8 genome sequences were compared with 34 published bacterial genomes, including closely related plant-associated bacteria not from crown galls. Homologous genes for virulence and opine anabolism were only present in the virulent Rhizobiaceae. In contrast, homologs of the opine catabolism genes were present in all strains including the nonvirulent members of the Rhizobiaceae and non-Rhizobiaceae. Gene neighborhood and sequence identity of the opine degradation cluster of virulent and nonvirulent strains together with the results of the opine utilization assay support the important role of opine utilization for cocolonization in crown galls, thereby shaping the crown gall community.
Modern lifestyle is often at odds with endogenously driven rhythmicity, which can lead to circadian disruption and metabolic syndrome. One signature for circadian disruption is a reduced or altered metabolite cycling in the circulating tissue reflecting the current metabolic status. Drosophila is a well-established model in chronobiology, but day-time dependent variations of transport metabolites in the fly circulation are poorly characterized. Here, we sampled fly hemolymph throughout the day and analyzed diacylglycerols (DGs), phosphoethanolamines (PEs) and phosphocholines (PCs) using LC-MS. In wild-type flies kept on sugar-only medium under a light-dark cycle, all transport lipid species showed a synchronized bimodal oscillation pattern with maxima at the beginning and end of the light phase which were impaired in period01 clock mutants. In wild-type flies under constant dark conditions, the oscillation became monophasic with a maximum in the middle of the subjective day. In strong support of clock-driven oscillations, levels of the targeted lipids peaked once in the middle of the light phase under time-restricted feeding independent of the time of food intake. When wild-type flies were reared on full standard medium, the rhythmic alterations of hemolymph lipid levels were greatly attenuated. Our data suggest that the circadian clock aligns daily oscillations of DGs, PEs, and PCs in the hemolymph to the anabolic siesta phase, with a strong influence of light on phase and modality.
Xylem hydraulic safety and efficiency are key traits determining tree fitness in a warmer and drier world. While numerous plant hydraulic studies have focused on branches, our understanding of root hydraulic functioning remains limited, although roots control water uptake, influence stomatal regulation and have commonly been considered as the most vulnerable organ along the hydraulic pathway.
We investigated 11 traits related to xylem safety and efficiency along the hydraulic pathway in four temperate broad-leaved tree species.
Continuous vessel tapering from coarse roots to stems and branches caused considerable reduction in hydraulic efficiency. Wood density was always lowest in roots, but did not decline linearly along the flow path. In contrast, xylem embolism resistance (P50) did not differ significantly between roots and branches, except for one species. The limited variation in xylem safety between organs did not adequately reflect the corresponding reductions in vessel diameter (by ~70%) and hydraulic efficiency (by ~85%). Although we did not observe any trade-off between xylem safety and specific conductivity, vessel diameter, vessel lumen fraction and wood density were related to embolism resistance, both across and partly within organs.
We conclude that coarse roots are not highly vulnerable to xylem embolism as commonly believed, indicating that hydraulic failure during soil drying might be restricted to fine roots.
The negative impact of juvenile undernourishment on adult behavior has been well reported for vertebrates, but relatively little is known about invertebrates. In honeybees, nutrition has long been known to affect task performance and timing of behavioral transitions. Whether and how a dietary restriction during larval development affects the task performance of adult honeybees is largely unknown. We raised honeybees in-vitro, varying the amount of a standardized diet (150 µl, 160 µl, 180 µl in total). Emerging adults were marked and inserted into established colonies. Behavioral performance of nurse bees and foragers was investigated and physiological factors known to be involved in the regulation of social organization were quantified. Surprisingly, adult honeybees raised under different feeding regimes did not differ in any of the behaviors observed. No differences were observed in physiological parameters apart from weight. Honeybees were lighter when undernourished (150 µl), while they were heavier under the overfed treatment (180 µl) compared to the control group raised under a normal diet (160 µl). These data suggest that dietary restrictions during larval development do not affect task performance or physiology in this social insect despite producing clear effects on adult weight. We speculate that possible effects of larval undernourishment might be compensated during the early period of adult life.
Plants do not have neurons but operate transmembrane ion channels and can get electrical excited by physical and chemical clues. Among them the Venus flytrap is characterized by its peculiar hapto-electric signaling. When insects collide with trigger hairs emerging the trap inner surface, the mechanical stimulus within the mechanosensory organ is translated into a calcium signal and an action potential (AP). Here we asked how the Ca\(^{2+}\) wave and AP is initiated in the trigger hair and how it is feed into systemic trap calcium-electrical networks. When Dionaea muscipula trigger hairs matures and develop hapto-electric excitability the mechanosensitive anion channel DmMSL10/FLYC1 and voltage dependent SKOR type Shaker K\(^{+}\) channel are expressed in the sheering stress sensitive podium. The podium of the trigger hair is interface to the flytrap’s prey capture and processing networks. In the excitable state touch stimulation of the trigger hair evokes a rise in the podium Ca2+ first and before the calcium signal together with an action potential travel all over the trap surface. In search for podium ion channels and pumps mediating touch induced Ca\(^{2+}\) transients, we, in mature trigger hairs firing fast Ca\(^{2+}\) signals and APs, found OSCA1.7 and GLR3.6 type Ca\(^{2+}\) channels and ACA2/10 Ca\(^{2+}\) pumps specifically expressed in the podium. Like trigger hair stimulation, glutamate application to the trap directly evoked a propagating Ca\(^{2+}\) and electrical event. Given that anesthetics affect K\(^+\) channels and glutamate receptors in the animal system we exposed flytraps to an ether atmosphere. As result propagation of touch and glutamate induced Ca\(^{2+}\) and AP long-distance signaling got suppressed, while the trap completely recovered excitability when ether was replaced by fresh air. In line with ether targeting a calcium channel addressing a Ca\(^{2+}\) activated anion channel the AP amplitude declined before the electrical signal ceased completely. Ether in the mechanosensory organ did neither prevent the touch induction of a calcium signal nor this post stimulus decay. This finding indicates that ether prevents the touch activated, glr3.6 expressing base of the trigger hair to excite the capture organ.
The evolution of the internal water transport system was a prerequisite for high plant productivity. In times of climate change, understanding the dependency of juvenile growth on xylem hydraulic physiology is therefore of high importance. Here, we explored various wood anatomical, hydraulic, and leaf morphological traits related to hydraulic safety and efficiency in three temperate broadleaved tree species (Acer pseudoplatanus, Betula pendula, and Sorbus aucuparia). We took advantage of a severe natural heat wave that resulted in different climatic growing conditions for even-aged plants from the same seed source growing inside a greenhouse and outside. Inside the greenhouse, the daily maximum vapour pressure deficit was on average 36% higher than outside during the growing seasons. Because of the higher atmospheric moisture stress, the biomass production differed up to 5.6-fold between both groups. Except for one species, a high productivity was associated with a high hydraulic efficiency caused by large xylem vessels and a large, supported leaf area. Although no safety-efficiency trade-off was observed, productivity was significantly related to P\(_{50}\) in two of the tree species but without revealing any clear pattern. A considerable plasticity in given traits was observed between both groups, with safety-related traits being more static while efficiency-related traits revealed a higher intra-specific plasticity. This was associated with other wood anatomical and leaf morphological adjustments. We confirm that a high hydraulic efficiency seems to be a prerequisite for a high biomass production, while our controversial results on the growth–xylem safety relationship confirm that safety-efficiency traits are decoupled and that their relationship with juvenile growth and water regime is species-specific.
Pivotal barrier properties of the hydrophobic plant cuticle covering aerial plant surfaces depend on its physicochemical composition. Among plant species and organs, compounds of this boundary layer between the plant interior and the environment vary considerably but cuticle-related studies comparing different organs from the same plant species are still scarce. Thus, this study focused on the cuticle profiles of Physalis peruviana, Physalis ixocarpa, Alkekengi officinarum, and Nicandra physalodes species. Inflated fruiting calyces enveloping fruits make Physalis, Alkekengi, and Nicandra highly recognizable genera among the Solanoideae subfamily. Although the inflation of fruiting calyces is well discussed in the literature still little is known about their post-floral functionalities. Cuticular composition, surface structure, and barrier function were examined and compared in fully expanded amphistomatous leaves, ripe astomatous fruits, and fully inflated hypostomatous fruiting calyces. Species- and organ-specific abundances of non-glandular and glandular trichomes revealed high structural diversity, covering not only abaxial and adaxial leaf surfaces but also fruiting calyx surfaces, whereas fruits were glabrous. Cuticular waxes, which limit non-stomatal transpiration, ranged from <1 μg cm\(^{−2}\) on P. peruviana fruiting calyces and N. physalodes fruits to 22 μg cm\(^{−2}\) on P. peruviana fruits. Very-long-chain aliphatic compounds, notably n-alkanes, iso-, and anteiso-branched alkanes, alkanols, alkanoic acids, and alkyl esters, dominated the cuticular wax coverages (≥86%). Diversity of cuticular wax patterns rose from leaves to fruiting calyces and peaked in fruits. The polymeric cutin matrix providing the structural framework for cuticular waxes was determined to range from 81 μg cm\(^{−2}\) for N. physalodes to 571 μg cm\(^{−2}\) for A. officinarum fruits. Cuticular transpiration barriers were highly efficient, with water permeabilities being ≤5 × 10\(^{−5}\) m s\(^{−1}\). Only the cuticular water permeability of N. physalodes fruits was 10 × 10\(^{−5}\) m s\(^{−1}\) leading to their early desiccation and fruits that easily split, whereas P. peruviana, P. ixocarpa, and A. officinarum bore fleshy fruits for extended periods after maturation. Regarding the functional significance, fruiting calyces establish a physicochemical shield that reduces water loss and enables fruit maturation within a protective microclimate, and promotes different seed dispersal strategies among plant species investigated.
Sphingolipid long-chain bases (LCBs) are building blocks for membrane-localized sphingolipids, and are involved in signal transduction pathways in plants. Elevated LCB levels are associated with the induction of programmed cell death and pathogen-derived toxin-induced cell death. Therefore, levels of free LCBs can determine survival of plant cells. To elucidate the contribution of metabolic pathways regulating high LCB levels, we applied the deuterium-labeled LCB D-erythro-sphinganine-d7 (D7-d18:0), the first LCB in sphingolipid biosynthesis, to Arabidopsis leaves and quantified labeled LCBs, LCB phosphates (LCB-Ps), and 14 abundant ceramide (Cer) species over time. We show that LCB D7-d18:0 is rapidly converted into the LCBs d18:0P, t18:0, and t18:0P. Deuterium-labeled ceramides were less abundant, but increased over time, with the highest levels detected for Cer(d18:0/16:0), Cer(d18:0/24:0), Cer(t18:0/16:0), and Cer(t18:0/22:0). A more than 50-fold increase of LCB-P levels after leaf incubation in LCB D7-d18:0 indicated that degradation of LCBs via LCB-Ps is important, and we hypothesized that LCB-P degradation could be a rate-limiting step to reduce high levels of LCBs. To functionally test this hypothesis, we constructed a transgenic line with dihydrosphingosine-1-phosphate lyase 1 (DPL1) under control of an inducible promotor. Higher expression of DPL1 significantly reduced elevated LCB-P and LCB levels induced by Fumonisin B1, and rendered plants more resistant against this fungal toxin. Taken together, we provide quantitative data on the contribution of major enzymatic pathways to reduce high LCB levels, which can trigger cell death. Specifically, we provide functional evidence that DPL1 can be a rate-limiting step in regulating high LCB levels.
For the treatment of large bone defects, the commonly used technique of autologous bone grafting presents several drawbacks and limitations. With the discovery of the bone-inducing capabilities of bone morphogenetic protein 2 (BMP2), several delivery techniques were developed and translated to clinical applications. Implantation of scaffolds containing adsorbed BMP2 showed promising results. However, off-label use of this protein-scaffold combination caused severe complications due to an uncontrolled release of the growth factor, which has to be applied in supraphysiological doses in order to induce bone formation. Here, we propose an alternative strategy that focuses on the covalent immobilization of an engineered BMP2 variant to biocompatible scaffolds. The new BMP2 variant harbors an artificial amino acid with a specific functional group, allowing a site-directed covalent scaffold functionalization. The introduced artificial amino acid does not alter BMP2′s bioactivity in vitro. When applied in vivo, the covalently coupled BMP2 variant induces the formation of bone tissue characterized by a structurally different morphology compared to that induced by the same scaffold containing ab-/adsorbed wild-type BMP2. Our results clearly show that this innovative technique comprises translational potential for the development of novel osteoinductive materials, improving safety for patients and reducing costs.
Xylem embolism resistance has been identified as a key trait with a causal relation to drought-induced tree mortality, but not much is known about its intra-specific trait variability (ITV) in dependence on environmental variation. We measured xylem safety and efficiency in 300 European beech (Fagus sylvatica L.) trees across 30 sites in Central Europe, covering a precipitation reduction from 886 to 522 mm year−1. A broad range of variables that might affect embolism resistance in mature trees, including climatic and soil water availability, competition, and branch age, were examined. The average P50 value varied by up to 1 MPa between sites. Neither climatic aridity nor structural variables had a significant influence on P50. However, P50 was less negative for trees with a higher soil water storage capacity, and positively related to branch age, while specific conductivity (Ks) was not significantly associated with either of these variables. The greatest part of the ITV for xylem safety and efficiency was attributed to random variability within populations. We conclude that the influence of site water availability on P50 and Ks is low in European beech, and that the high degree of within-population variability for P50, partly due to variation in branch age, hampers the identification of a clear environmental signal.
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.
In vitro rearing of honeybee larvae is an established method that enables exact control and monitoring of developmental factors and allows controlled application of pesticides or pathogens. However, only a few studies have investigated how the rearing method itself affects the behavior of the resulting adult honeybees. We raised honeybees in vitro according to a standardized protocol: marking the emerging honeybees individually and inserting them into established colonies. Subsequently, we investigated the behavioral performance of nurse bees and foragers and quantified the physiological factors underlying the social organization. Adult honeybees raised in vitro differed from naturally reared honeybees in their probability of performing social tasks. Further, in vitro-reared bees foraged for a shorter duration in their life and performed fewer foraging trips. Nursing behavior appeared to be unaffected by rearing condition. Weight was also unaffected by rearing condition. Interestingly, juvenile hormone titers, which normally increase strongly around the time when a honeybee becomes a forager, were significantly lower in three- and four-week-old in vitro bees. The effects of the rearing environment on individual sucrose responsiveness and lipid levels were rather minor. These data suggest that larval rearing conditions can affect the task performance and physiology of adult bees despite equal weight, pointing to an important role of the colony environment for these factors. Our observations of behavior and metabolic pathways offer important novel insight into how the rearing environment affects adult honeybees.
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.
Protein purification is the vital basis to study the function, structure and interaction of proteins. Widely used methods are affinity chromatography-based purifications, which require different chromatography columns and harsh conditions, such as acidic pH and/or adding imidazole or high salt concentration, to elute and collect the purified proteins. Here we established an easy and fast purification method for soluble proteins under mild conditions, based on the light-induced protein dimerization system improved light-induced dimer (iLID), which regulates protein binding and release with light. We utilize the biological membrane, which can be easily separated by centrifugation, as the port to anchor the target proteins. In Xenopus laevis oocyte and Escherichia coli, the blue light-sensitive part of iLID, AsLOV2-SsrA, was targeted to the plasma membrane by different membrane anchors. The other part of iLID, SspB, was fused with the protein of interest (POI) and expressed in the cytosol. The SspB-POI can be captured to the membrane fraction through light-induced binding to AsLOV2-SsrA and then released purely to fresh buffer in the dark after simple centrifugation and washing. This method, named mem-iLID, is very flexible in scale and economic. We demonstrate the quickly obtained yield of two pure and fully functional enzymes: a DNA polymerase and a light-activated adenylyl cyclase. Furthermore, we also designed a new SspB mutant for better dissociation and less interference with the POI, which could potentially facilitate other optogenetic manipulations of protein–protein interaction.
Optogenetics was developed in the field of neuroscience and is most commonly using light-sensitive rhodopsins to control the neural activities. Lately, we have expanded this technique into plant science by co-expression of a chloroplast-targeted β-carotene dioxygenase and an improved anion channelrhodopsin GtACR1 from the green alga Guillardia theta. The growth of Nicotiana tabacum pollen tube can then be manipulated by localized green light illumination. To extend the application of analogous optogenetic tools in the pollen tube system, we engineered another two ACRs, GtACR2, and ZipACR, which have different action spectra, light sensitivity and kinetic features, and characterized them in Xenopus laevis oocytes, Nicotiana benthamiana leaves and N. tabacum pollen tubes. We found that the similar molecular engineering method used to improve GtACR1 also enhanced GtACR2 and ZipACR performance in Xenopus laevis oocytes. The ZipACR1 performed in N. benthamiana mesophyll cells and N. tabacum pollen tubes with faster kinetics and reduced light sensitivity, allowing for optogenetic control of anion fluxes with better temporal resolution. The reduced light sensitivity would potentially facilitate future application in plants, grown under low ambient white light, combined with an optogenetic manipulation triggered by stronger green light.
Background
Microbial rhodopsins vary in their chemical properties, from light sensitive ion transport to different enzymatic activities. Recently, a novel family of two-component Cyclase (rhod)opsins (2c-Cyclop) from the green algae Chlamydomonas reinhardtii and Volvox carteri was characterized, revealing a light-inhibited guanylyl cyclase (GC) activity. More genes similar to 2c-Cyclop exist in algal genomes, but their molecular and physiological functions remained uncharacterized.
Results
Chlamyopsin-5 (Cop5) from C. reinhardtii is related to Cr2c-Cyclop1 (Cop6) and can be expressed in Xenopus laevis oocytes, but shows no GC activity. Here, we exchanged parts of Cop5 with the corresponding ones of Cr2c-Cyclop1. When exchanging the opsin part of Cr2c-Cyclop1 with that of Cop5, we obtained a bi-stable guanylyl cyclase (switch-Cyclop1) whose activity can be switched by short light flashes. The GC activity of switch-Cyclop1 is increased for hours by a short 380 nm illumination and switched off (20-fold decreased) by blue or green light. switch-Cyclop1 is very light-sensitive and can half-maximally be activated by ~ 150 photons/nm2 of 380 nm (~ 73 J/m2) or inhibited by ~ 40 photons/nm\(^2\) of 473 nm (~ 18 J/m\(^2\)).
Conclusions
This engineered guanylyl cyclase is the first light-switchable enzyme for cGMP level regulation. Light-regulated cGMP production with high light-sensitivity is a promising technique for the non-invasive investigation of the effects of cGMP signaling in many different tissues.
Whereas the role of calcium ions (Ca\(^{2+}\)) in plant signaling is well studied, the physiological significance of pH‐changes remains largely undefined.
Here we developed CapHensor, an optimized dual‐reporter for simultaneous Ca\(^{2+}\) and pH ratio‐imaging and studied signaling events in pollen tubes (PTs), guard cells (GCs), and mesophyll cells (MCs). Monitoring spatio‐temporal relationships between membrane voltage, Ca\(^{2+}\)‐ and pH‐dynamics revealed interconnections previously not described.
In tobacco PTs, we demonstrated Ca\(^{2+}\)‐dynamics lag behind pH‐dynamics during oscillatory growth, and pH correlates more with growth than Ca\(^{2+}\). In GCs, we demonstrated abscisic acid (ABA) to initiate stomatal closure via rapid cytosolic alkalization followed by Ca2+ elevation. Preventing the alkalization blocked GC ABA‐responses and even opened stomata in the presence of ABA, disclosing an important pH‐dependent GC signaling node. In MCs, a flg22‐induced membrane depolarization preceded Ca2+‐increases and cytosolic acidification by c. 2 min, suggesting a Ca\(^{2+}\)/pH‐independent early pathogen signaling step. Imaging Ca2+ and pH resolved similar cytosol and nuclear signals and demonstrated flg22, but not ABA and hydrogen peroxide to initiate rapid membrane voltage‐, Ca\(^{2+}\)‐ and pH‐responses.
We propose close interrelation in Ca\(^{2+}\)‐ and pH‐signaling that is cell type‐ and stimulus‐specific and the pH having crucial roles in regulating PT growth and stomata movement.
Plant stress signalling involves bursts of reactive oxygen species (ROS), which can be mimicked by the application of acute pulses of ozone. Such ozone-pulses inhibit photosynthesis and trigger stomatal closure in a few minutes, but the signalling that underlies these responses remains largely unknown.
We measured changes in Arabidopsis thaliana gas exchange after treatment with acute pulses of ozone and set up a system for simultaneous measurement of membrane potential and cytosolic calcium with the fluorescent reporter R-GECO1.
We show that within 1 min, prior to stomatal closure, O\(_{3}\) triggered a drop in whole-plant CO\(_{2}\) uptake. Within this early phase, O\(_{3}\) pulses (200–1000 ppb) elicited simultaneous membrane depolarization and cytosolic calcium increase, whereas these pulses had no long-term effect on either stomatal conductance or photosynthesis. In contrast, pulses of 5000 ppb O\(_{3}\) induced cell death, systemic Ca\(^{2+}\) signals and an irreversible drop in stomatal conductance and photosynthetic capacity.
We conclude that mesophyll cells respond to ozone in a few seconds by distinct pattern of plasma membrane depolarizations accompanied by an increase in the cytosolic calcium ion (Ca\(^{2+}\)) level. These responses became systemic only at very high ozone concentrations. Thus, plants have rapid mechanism to sense and discriminate the strength of ozone signals.
Cytosolic calcium signals are evoked by a large variety of biotic and abiotic stimuli and play an important role in cellular and long distance signalling in plants. While the function of the plasma membrane in cytosolic Ca\(^{2+}\) signalling has been intensively studied, the role of the vacuolar membrane remains elusive.
A newly developed vacuolar voltage clamp technique was used in combination with live-cell imaging, to study the role of the vacuolar membrane in Ca\(^{2+}\) and pH homeostasis of bulging root hair cells of Arabidopsis.
Depolarisation of the vacuolar membrane caused a rapid increase in the Ca\(^{2+}\) concentration and alkalised the cytosol, while hyperpolarisation led to the opposite responses.
The relationship between the vacuolar membrane potential, the cytosolic pH and Ca2+ concentration suggests that a vacuolar H\(^{+}\)/Ca\(^{2+}\) exchange mechanism plays a central role in cytosolic Ca2+ homeostasis. Mathematical modelling further suggests that the voltage-dependent vacuolar Ca\(^{2+}\) homeostat could contribute to calcium signalling when coupled to a recently discovered K\(^{+}\) channel-dependent module for electrical excitability of the vacuolar membrane.
Background
While leaves are far more accessible for analysing plant defences, roots are hidden in the soil, leading to difficulties in studying soil-borne interactions. Inoculation strategies for infecting model plants with model root pathogens are described in the literature, but it remains demanding to obtain a methodological overview. To address this challenge, this study uses the model root pathogen Verticillium longisporum on Arabidopsis thaliana host plants and provides recommendations for selecting appropriate infection systems to investigate how plants cope with root pathogens.
Results
A novel root infection system is introduced, while two existing ones are precisely described and optimized. Step-by-step protocols are presented and accompanied by pathogenicity tests, transcriptional analyses of indole-glucosinolate marker genes and independent confirmations using reporter constructs. Advantages and disadvantages of each infection system are assessed. Overall, the results validate the importance of indole-glucosinolates as secondary metabolites that limit the Verticillium propagation in its host plant.
Conclusion
Detailed assistances on studying host defence strategies and responses against V. longisporum is provided. Furthermore, other soil-borne microorganisms (e.g., V. dahliae) or model plants, such as economically important oilseed rape and tomato, can be introduced in the infection systems described. Hence, these proven manuals can support finding a root infection system for your specific research questions to further decipher root-microbe interactions.
Epidermal fragments enriched in guard cells (GCs) were isolated from the halophyte quinoa (Chenopodium quinoa Wild.) species, and the response at the proteome level was studied after salinity treatment of 300 mM NaCl for 3 weeks. In total, 2147 proteins were identified, of which 36% were differentially expressed in response to salinity stress in GCs. Up and downregulated proteins included signaling molecules, enzyme modulators, transcription factors and oxidoreductases. The most abundant proteins induced by salt treatment were desiccation-responsive protein 29B (50-fold), osmotin-like protein OSML13 (13-fold), polycystin-1, lipoxygenase, alpha-toxin, and triacylglycerol lipase (PLAT) domain-containing protein 3-like (eight-fold), and dehydrin early responsive to dehydration (ERD14) (eight-fold). Ten proteins related to the gene ontology term “response to ABA” were upregulated in quinoa GC; this included aspartic protease, phospholipase D and plastid-lipid-associated protein. Additionally, seven proteins in the sucrose–starch pathway were upregulated in the GC in response to salinity stress, and accumulation of tryptophan synthase and L-methionine synthase (enzymes involved in the amino acid biosynthesis) was observed. Exogenous application of sucrose and tryptophan, L-methionine resulted in reduction in stomatal aperture and conductance, which could be advantageous for plants under salt stress. Eight aspartic proteinase proteins were highly upregulated in GCs of quinoa, and exogenous application of pepstatin A (an inhibitor of aspartic proteinase) was accompanied by higher oxidative stress and extremely low stomatal aperture and conductance, suggesting a possible role of aspartic proteinase in mitigating oxidative stress induced by saline conditions.
Guard cells control the aperture of plant stomata, which are crucial for global fluxes of CO\(_2\) and water. In turn, guard cell anion channels are seen as key players for stomatal closure, but is activation of these channels sufficient to limit plant water loss? To answer this open question, we used an optogenetic approach based on the light-gated anion channelrhodopsin 1 (GtACR1). In tobacco guard cells that express GtACR1, blue- and green-light pulses elicit Cl\(^-\) and NO\(_3\)\(^-\) currents of -1 to -2 nA. The anion currents depolarize the plasma membrane by 60 to 80 mV, which causes opening of voltage-gated K+ channels and the extrusion of K+. As a result, continuous stimulation with green light leads to loss of guard cell turgor and closure of stomata at conditions that provoke stomatal opening in wild type. GtACR1 optogenetics thus provides unequivocal evidence that opening of anion channels is sufficient to close stomata.
Simple Summary
Abiotic and biotic stress conditions result in profound changes in plant lipid metabolism. Vegetable oil consists of triacylglycerols, which are important energy and carbon storage compounds in seeds of various plant species. These compounds are also present in vegetative tissue, and levels have been reported to increase with different abiotic stresses in leaves. This work shows that triacylglycerols accumulate in roots and in distal, non-treated leaves upon treatment with a fungal pathogen or lipopolysaccharide (a common bacterial-derived elicitor in animals and plants). Treatment of leaves with a bacterial pathogen or a bacterial effector molecule results in triacylglycerol accumulation in leaves, but not systemically in roots. These results suggest that elicitor molecules are sufficient to induce an increase in triacylglycerol levels, and that unidirectional long-distance signaling from roots to leaves is involved in pathogen and elicitor-induced triacylglycerol accumulation.
Abstract
Interaction of plants with the environment affects lipid metabolism. Changes in the pattern of phospholipids have been reported in response to abiotic stress, particularly accumulation of triacylglycerols, but less is known about the alteration of lipid metabolism in response to biotic stress and leaves have been more intensively studied than roots. This work investigates the levels of lipids in roots as well as leaves of Arabidopsis thaliana in response to pathogens and elicitor molecules by UPLC-TOF-MS. Triacylglycerol levels increased in roots and systemically in leaves upon treatment of roots with the fungus Verticillium longisporum. Upon spray infection of leaves with the bacterial pathogen Pseudomonas syringae, triacylglycerols accumulated locally in leaves but not in roots. Treatment of roots with a bacterial lipopolysaccharide elicitor induced a strong triacylglycerol accumulation in roots and leaves. Induction of the expression of the bacterial effector AVRRPM1 resulted in a dramatic increase of triacylglycerol levels in leaves, indicating that elicitor molecules are sufficient to induce accumulation of triacylglycerols. These results give insight into local and systemic changes to lipid metabolism in roots and leaves in response to biotic stresses.
Climate change is increasing the frequency and intensity of warming and drought periods around the globe, currently representing a threat to many plant species. Understanding the resistance and resilience of plants to climate change is, therefore, urgently needed. As date palm (Phoenix dactylifera) evolved adaptation mechanisms to a xeric environment and can tolerate large diurnal and seasonal temperature fluctuations, we studied the protein expression changes in leaves, volatile organic compound emissions, and photosynthesis in response to variable growth temperatures and soil water deprivation. Plants were grown under controlled environmental conditions of simulated Saudi Arabian summer and winter climates challenged with drought stress. We show that date palm is able to counteract the harsh conditions of the Arabian Peninsula by adjusting the abundances of proteins related to the photosynthetic machinery, abiotic stress and secondary metabolism. Under summer climate and water deprivation, these adjustments included efficient protein expression response mediated by heat shock proteins and the antioxidant system to counteract reactive oxygen species formation. Proteins related to secondary metabolism were downregulated, except for the P. dactylifera isoprene synthase (PdIspS), which was strongly upregulated in response to summer climate and drought. This study reports, for the first time, the identification and functional characterization of the gene encoding for PdIspS, allowing future analysis of isoprene functions in date palm under extreme environments. Overall, the current study shows that reprogramming of the leaf protein profiles confers the date palm heat- and drought tolerance. We conclude that the protein plasticity of date palm is an important mechanism of molecular adaptation to environmental fluctuations.
Key message
Mobile laser scanning and geometrical analysis revealed relationships between tree geometry and seed dispersal mechanism, latitude of origin, as well as growth.
Abstract
The structure and dynamics of a forest are defined by the architecture and growth patterns of its individual trees. In turn, tree architecture and growth result from the interplay between the genetic building plans and environmental factors. We set out to investigate whether (1) latitudinal adaptations of the crown shape occur due to characteristic solar elevation angles at a species’ origin, (2) architectural differences in trees are related to seed dispersal strategies, and (3) tree architecture relates to tree growth performance. We used mobile laser scanning (MLS) to scan 473 trees and generated three-dimensional data of each tree. Tree architectural complexity was then characterized by fractal analysis using the box-dimension approach along with a topological measure of the top heaviness of a tree. The tree species studied originated from various latitudinal ranges, but were grown in the same environmental settings in the arboretum. We found that trees originating from higher latitudes had significantly less top-heavy geometries than those from lower latitudes. Therefore, to a certain degree, the crown shape of tree species seems to be determined by their original habitat. We also found that tree species with wind-dispersed seeds had a higher structural complexity than those with animal-dispersed seeds (p < 0.001). Furthermore, tree architectural complexity was positively related to the growth performance of the trees (p < 0.001). We conclude that the use of 3D data from MLS in combination with geometrical analysis, including fractal analysis, is a promising tool to investigate tree architecture.
Fungal endophytes of the genus Epichloë live symbiotically in cool season grass species and can produce alkaloids toxic to insects and vertebrates, yet reports of intoxication of grazing animals have been rare in Europe in contrast to overseas. However, due to the beneficial resistance traits observed in Epichloë infected grasses, the inclusion of Epichloë in seed mixtures might become increasingly advantageous. Despite the toxicity of fungal alkaloids, European seed mixtures are rarely tested for Epichloë infection and their infection status is unknown for consumers. In this study, we tested 24 commercially available seed mixtures for their infection rates with Epichloë endophytes and measured the concentrations of the alkaloids ergovaline, lolitrem B, paxilline, and peramine. We detected Epichloë infections in six seed mixtures, and four contained vertebrate and insect toxic alkaloids typical for Epichloë festucae var. lolii infecting Lolium perenne. As Epichloë infected seed mixtures can harm livestock, when infected grasses become dominant in the seeded grasslands, we recommend seed producers to test and communicate Epichloë infection status or avoiding Epichloë infected seed mixtures.
The plant hormone jasmonoyl-isoleucine (JA-Ile) is an important regulator of plant growth and defense in response to various biotic and abiotic stress cues. Under our experimental conditions, JA-Ile levels increased approximately seven-fold in NaCl-treated Arabidopsis thaliana roots. Although these levels were around 1000-fold lower than in wounded leaves, genes of the JA-Ile signaling pathway were induced by a factor of 100 or more. Induction was severely compromised in plants lacking the JA-Ile receptor CORONATINE INSENSITIVE 1 or enzymes required for JA-Ile biosynthesis. To explain efficient gene expression at very low JA-Ile levels, we hypothesized that salt-induced expression of the JA/JA-Ile transporter JAT1/AtABCG16 would lead to increased nuclear levels of JA-Ile. However, mutant plants with different jat1 alleles were similar to wild-type ones with respect to salt-induced gene expression. The mechanism that allows COI1-dependent gene expression at very low JA-Ile levels remains to be elucidated.
Plant transpiration is a key element in the hydrological cycle. Widely used methods for its assessment comprise sap flux techniques for whole-plant transpiration and porometry for leaf stomatal conductance. Recently emerging approaches based on surface temperatures and a wide range of machine learning techniques offer new possibilities to quantify transpiration. The focus of this study was to predict sap flux and leaf stomatal conductance based on drone-recorded and meteorological data and compare these predictions with in-situ measured transpiration. To build the prediction models, we applied classical statistical approaches and machine learning algorithms. The field work was conducted in an oil palm agroforest in lowland Sumatra. Random forest predictions yielded the highest congruence with measured sap flux (r\(^2\) = 0.87 for trees and r\(^2\) = 0.58 for palms) and confidence intervals for intercept and slope of a Passing-Bablok regression suggest interchangeability of the methods. Differences in model performance are indicated when predicting different tree species. Predictions for stomatal conductance were less congruent for all prediction methods, likely due to spatial and temporal offsets of the measurements. Overall, the applied drone and modelling scheme predicts whole-plant transpiration with high accuracy. We conclude that there is large potential in machine learning approaches for ecological applications such as predicting transpiration.
While much research has addressed the aboveground response of trees to climate warming and related water shortage, not much is known about the drought sensitivity of the fine root system, in particular of mature trees. This study investigates the response of topsoil (0–10 cm) fine root biomass (FRB), necromass (FRN), and fine root morphology of five temperate broadleaf tree species (Acer platanoides L., Carpinus betulus L., Fraxinus excelsior L., Quercus petraea (Matt.) Liebl., Tilia cordata Mill.) to a reduction in water availability, combining a precipitation gradient study (nine study sites; mean annual precipitation (MAP): 920–530 mm year\(^{−1}\)) with the comparison of a moist period (average spring conditions) and an exceptionally dry period in the summer of the subsequent year. The extent of the root necromass/biomass (N/B) ratio increase was used as a measure of the species’ belowground sensitivity to water deficits. We hypothesized that the N/B ratio increases with long-term (precipitation gradient) and short-term reductions (moist vs. dry period) of water availability, while FRB changes only a little. In four of the five species (exception: A. platanoides), FRB did not change with a reduction in MAP, whereas FRN and N/B ratio increased toward the dry sites under ample water supply (exception: Q. petraea). Q. petraea was also the only species not to reduce root tip frequency after summer drought. Different slopes of the N/B ratio-MAP relation similarly point at a lower belowground drought sensitivity of Q. petraea than of the other species. After summer drought, all species lost the MAP dependence of the N/B ratio. Thus, fine root mortality increased more at the moister than the drier sites, suggesting a generally lower belowground drought sensitivity of the drier stands. We conclude that the five species differ in their belowground drought response. Q. petraea follows the most conservative soil exploration strategy with a generally smaller FRB and more drought-tolerant fine roots, as it maintains relatively constant FRB, FRN, and morphology across spatial and temporal dimensions of soil water deficits.
Mycotoxins in agriculturally used plants can cause intoxication in animals and can lead to severe financial losses for farmers. The endophytic fungus Epichloë festucae var. lolii living symbiotically within the cool season grass species Lolium perenne can produce vertebrate and invertebrate toxic alkaloids. Hence, an exact quantitation of alkaloid concentrations is essential to determine intoxication risk for animals. Many studies use different methods to detect alkaloid concentrations, which complicates the comparability. In this study, we showed that alkaloid concentrations of individual plants exceeded toxicity thresholds on real world grasslands in Germany, but not on the population level. Alkaloid concentrations on five German grasslands with high alkaloid levels peaked in summer but were also below toxicity thresholds on population level. Furthermore, we showed that alkaloid concentrations follow the same seasonal trend, regardless of whether plant fresh or dry weight was used, in the field and in a common garden study. However, alkaloid concentrations were around three times higher when detected with dry weight. Finally, we showed that alkaloid concentrations can additionally be biased to different alkaloid detection methods. We highlight that toxicity risks should be analyzed using plant dry weight, but concentration trends of fresh weight are reliable.
Background
The plant endophytic fungus Serendipita indica colonizes roots of a wide range of plant species and can enhance growth and stress resistance of these plants. Due to its ease of axenic cultivation and its broad host plant range including the model plant Arabidopsis thaliana and numerous crop plants, it is widely used as a model fungus to study beneficial fungus-root interactions. In addition, it was suggested to be utilized for commercial applications, e.g. to enhance yield in barley and other species. To produce inoculum, S. indica is mostly cultivated in a complex Hill-Kafer medium (CM medium), however, growth in this medium is slow, and yield of chlamydospores, which are often used for plant root inoculation, is relatively low.
Results
We tested and optimized a simple vegetable juice-based medium for an enhanced yield of fungal inoculum. The described vegetable juice (VJ) medium is based on commercially available vegetable juice and is easy to prepare. VJ medium was superior to the currently used CM medium with respect to biomass production in liquid medium and hyphal growth on agar plates. Using solid VJ medium supplemented with sucrose (VJS), a high amount of chlamydospores developed already after 8 days of cultivation, producing significantly more spores than on CM medium. Use of VJ medium is not restricted to S. indica, as it also supported growth of two pathogenic fungi often used in plant pathology experiments: the ascomycete Fusarium graminearum, the causal agent of Fusarium head blight disease on wheat and barley, and Verticillium longisporum, the causal agent of verticillium wilt.
Conclusions
The described VJ medium is recommended for streamlined and efficient production of inoculum for the plant endophytic fungus Serendipita indica and might prove superior for the propagation of other fungi for research purposes.
The fruit fly Drosophila is a prime model in circadian research, but still little is known about its circadian regulation of metabolism. Daily rhythmicity in levels of several metabolites has been found, but knowledge about hydrophobic metabolites is limited. We here compared metabolite levels including lipids between period\(^{01}\) (per\(^{01}\)) clock mutants and Canton-S wildtype (WT\(_{CS}\)) flies in an isogenic and non-isogenic background using LC–MS. In the non-isogenic background, metabo-lites with differing levels comprised essential amino acids, kynurenines, pterinates, glycero(phospho)lipids, and fatty acid esters. Notably, detectable diacylglycerols (DAG) and acylcarnitines (AC), involved in lipid metabolism, showed lower levels in per\(^{01}\) mutants. Most of these differences disappeared in the isogenic background, yet the level differences for AC as well as DAG were consistent for fly bodies. AC levels were dependent on the time of day in WTCS in phase with food consumption under LD conditions, while DAGs showed weak daily oscillations. Two short-chain ACs continued to cycle even in constant darkness. per\(^{01}\) mutants in LD showed no or very weak diel AC oscillations out of phase with feeding activity. The low levels of DAGs and ACs in per\(^{01}\) did not correlate with lower total food consumption, body mass or weight. Clock mutant flies showed higher sensitivity to starvation independent of their background-dependent activity level. Our results suggest that neither feeding, energy storage nor mobilisation is significantly affected in per\(^{01}\) mutants, but point towards impaired mitochondrial activity, supported by upregulation of the mitochondrial stress marker 4EBP in the clock mutants
The origins of multicellular physiology are tied to evolution of gene expression. Genes can shift expression as organisms evolve, but how ancestral expression influences altered descendant expression is not well understood. To examine this, we amalgamate 1,903 RNA-seq datasets from 182 research projects, including 6 organs in 21 vertebrate species. Quality control eliminates project-specific biases, and expression shifts are reconstructed using gene-family-wise phylogenetic Ornstein-Uhlenbeck models. Expression shifts following gene duplication result in more drastic changes in expression properties than shifts without gene duplication. The expression properties are tightly coupled with protein evolutionary rate, depending on whether and how gene duplication occurred. Fluxes in expression patterns among organs are nonrandom, forming modular connections that are reshaped by gene duplication. Thus, if expression shifts, ancestral expression in some organs induces a strong propensity for expression in particular organs in descendants. Regardless of whether the shifts are adaptive or not, this supports a major role for what might be termed preadaptive pathways of gene expression evolution.
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.
In Brassicaceae, tissue damage triggers the mustard oil bomb i.e., activates the degradation of glucosinolates by myrosinases leading to a rapid accumulation of isothiocyanates at the site of damage. Isothiocyanates are reactive electrophilic species (RES) known to covalently bind to thiols in proteins and glutathione, a process that is not only toxic to herbivores and microbes but can also cause cell death of healthy plant tissues. Previously, it has been shown that subtoxic isothiocyanate concentrations can induce transcriptional reprogramming in intact plant cells. Glutathione depletion by RES leading to breakdown of the redox potential has been proposed as a central and common RES signal transduction mechanism. Using transcriptome analyses, we show that after exposure of Arabidopsis seedlings (grown in liquid culture) to subtoxic concentrations of sulforaphane hundreds of genes were regulated without depletion of the cellular glutathione pool. Heat shock genes were among the most highly up-regulated genes and this response was found to be dependent on the canonical heat shock factors A1 (HSFA1). HSFA1-deficient plants were more sensitive to isothiocyanates than wild type plants. Moreover, pretreatment of Arabidopsis seedlings with subtoxic concentrations of isothiocyanates increased resistance against exposure to toxic levels of isothiocyanates and, hence, may reduce the autotoxicity of the mustard oil bomb by inducing cell protection mechanisms.
Soil salinity is a major environmental constraint affecting crop growth and threatening global food security. Plants adapt to salinity by optimizing the performance of stomata. Stomata are formed by two guard cells (GCs) that are morphologically and functionally distinct from the other leaf cells. These microscopic sphincters inserted into the wax-covered epidermis of the shoot balance CO\(_2\) intake for photosynthetic carbon gain and concomitant water loss. In order to better understand the molecular mechanisms underlying stomatal function under saline conditions, we used proteomics approach to study isolated GCs from the salt-tolerant sugar beet species. Of the 2088 proteins identified in sugar beet GCs, 82 were differentially regulated by salt treatment. According to bioinformatics analysis (GO enrichment analysis and protein classification), these proteins were involved in lipid metabolism, cell wall modification, ATP biosynthesis, and signaling. Among the significant differentially abundant proteins, several proteins classified as “stress proteins” were upregulated, including non-specific lipid transfer protein, chaperone proteins, heat shock proteins, inorganic pyrophosphatase 2, responsible for energized vacuole membrane for ion transportation. Moreover, several antioxidant enzymes (peroxide, superoxidase dismutase) were highly upregulated. Furthermore, cell wall proteins detected in GCs provided some evidence that GC walls were more flexible in response to salt stress. Proteins such as L-ascorbate oxidase that were constitutively high under both control and high salinity conditions may contribute to the ability of sugar beet GCs to adapt to salinity by mitigating salinity-induced oxidative stress.
The carbohydrate D-glucose is the main source of energy in living organisms. In contrast to animals, as well as most fungi, bacteria, and archaea, plants are capable to synthesize a surplus of sugars characterizing them as autothrophic organisms. Thus, plants are de facto the source of all food on earth, either directly or indirectly via feed to livestock. Glucose is stored as polymeric glucan, in animals as glycogen and in plants as starch. Despite serving a general source for metabolic energy and energy storage, glucose is the main building block for cellulose synthesis and represents the metabolic starting point of carboxylate- and amino acid synthesis. Finally yet importantly, glucose functions as signalling molecule conveying the plant metabolic status for adjustment of growth, development, and survival. Therefore, cell-to-cell and long-distance transport of photoassimilates/sugars throughout the plant body require the fine-tuned activity of sugar transporters facilitating the transport across membranes. The functional plant counterparts of the animal sodium/glucose transporters (SGLTs) are represented by the proton-coupled sugar transport proteins (STPs) of the plant monosaccharide transporter(-like) family (MST). In the framework of this special issue on “Glucose Transporters in Health and Disease,” this review gives an overview of the function and structure of plant STPs in comparison to the respective knowledge obtained with the animal Na+-coupled glucose transporters (SGLTs).
Studying how cambial age and axial height affects wood anatomical traits may improve our understanding of xylem hydraulics, heartwood formation and axial growth. Radial strips were collected from six different heights (0–11.3 m) along the main trunk of three Manchurian catalpa (Catalpa bungei) trees, yielding 88 samples. In total, thirteen wood anatomical vessel and fiber traits were observed usinglight microscopy (LM) and scanning electron microscopy (SEM), and linear models were used to analyse the combined effect of axial height, cambial age and their interaction. Vessel diameter differed by about one order of magnitude between early- and latewood, and increased significantly with both cambial age and axial height in latewood, while it was positively affected by cambial age and independent of height in earlywood. Vertical position further had a positive effect on earlywood vessel density, and negative effects on fibre wall thickness, wall thickness to diameter ratio and length. Cambial age had positive effects on the pit membrane diameter and vessel element length, while the annual diameter growth decreased with both cambial age and axial position. In contrast, early- and latewood fiber diameter were unaffected by both cambial age and axial height. We further observed an increasing amount of tyloses from sapwood to heartwood, accompanied by an increase of warty layers and amorphous deposits on cell walls, bordered pit membranes and pit apertures. This study highlights the significant effects of cambial age and vertical position on xylem anatomical traits, and confirms earlier work that cautions to take into account xylem spatial position when interpreting wood anatomical structures, and thus, xylem hydraulic functioning.
Bone Morphogenetic Proteins (BMPs) together with the Growth and Differentiation Factors (GDFs) form the largest subgroup of the Transforming Growth Factor (TGF)β family and represent secreted growth factors, which play an essential role in many aspects of cell communication in higher organisms. As morphogens they exert crucial functions during embryonal development, but are also involved in tissue homeostasis and regeneration in the adult organism. Their involvement in maintenance and repair processes of various tissues and organs made these growth factors highly interesting targets for novel pharmaceutical applications in regenerative medicine. A hallmark of the TGFβ protein family is that all of the more than 30 growth factors identified to date signal by binding and hetero-oligomerization of a very limited set of transmembrane serine-threonine kinase receptors, which can be classified into two subgroups termed type I and type II. Only seven type I and five type II receptors exist for all 30plus TGFβ members suggesting a pronounced ligand-receptor promiscuity. Indeed, many TGFβ ligands can bind the same type I or type II receptor and a particular receptor of either subtype can usually interact with and bind various TGFβ ligands. The possible consequence of this ligand-receptor promiscuity is further aggravated by the finding that canonical TGFβ signaling of all family members seemingly results in the activation of just two distinct signaling pathways, that is either SMAD2/3 or SMAD1/5/8 activation. While this would implicate that different ligands can assemble seemingly identical receptor complexes that activate just either one of two distinct pathways, in vitro and in vivo analyses show that the different TGFβ members exert quite distinct biological functions with high specificity. This discrepancy indicates that our current view of TGFβ signaling initiation just by hetero-oligomerization of two receptor subtypes and transduction via two main pathways in an on-off switch manner is too simplified. Hence, the signals generated by the various TGFβ members are either quantitatively interpreted using the subtle differences in their receptor-binding properties leading to ligand-specific modulation of the downstream signaling cascade or additional components participating in the signaling activation complex allow diversification of the encoded signal in a ligand-dependent manner at all cellular levels. In this review we focus on signal specification of TGFβ members, particularly of BMPs and GDFs addressing the role of binding affinities, specificities, and kinetics of individual ligand-receptor interactions for the assembly of specific receptor complexes with potentially distinct signaling properties.
Metabolomic profiling of different Premna odorata Blanco (Lamiaceae) organs, bark, wood, young stems, flowers, and fruits dereplicated 20, 20, 10, 20, and 20 compounds, respectively, using LC–HRESIMS. The identified metabolites (1–34) belonged to different chemical classes, including iridoids, flavones, phenyl ethanoids, and lignans. A phytochemical investigation of P. odorata bark afforded one new tetrahydrofurofuran lignan, 4β-hydroxyasarinin 35, along with fourteen known compounds. The structure of the new compound was confirmed using extensive 1D and 2D NMR, and HRESIMS analyses. A cytotoxic investigation of compounds 35–38 against the HL-60, HT-29, and MCF-7 cancer cell lines, using the MTT assay showed that compound 35 had cytotoxic effects against HL-60 and MCF-7 with IC50 values of 2.7 and 4.2 µg/mL, respectively. A pharmacophore map of compounds 35 showed two hydrogen bond acceptor (HBA) aligning the phenoxy oxygen atoms of benzodioxole moieties, two aromatic ring features vectored on the two phenyl rings, one hydrogen bond donor (HBD) feature aligning the central hydroxyl group and thirteen exclusion spheres which limit the boundaries of sterically inaccessible regions of the target’s active site.
(1) Background: After the discovery and application of Chlamydomonas reinhardtii channelrhodopsins, the optogenetic toolbox has been greatly expanded with engineered and newly discovered natural channelrhodopsins. However, channelrhodopsins of higher Ca\(^{2+}\) conductance or more specific ion permeability are in demand. (2) Methods: In this study, we mutated the conserved aspartate of the transmembrane helix 4 (TM4) within Chronos and PsChR and compared them with published ChR2 aspartate mutants. (3) Results: We found that the ChR2 D156H mutant (XXM) showed enhanced Na\(^+\) and Ca\(^{2+}\) conductance, which was not noticed before, while the D156C mutation (XXL) influenced the Na\(^+\) and Ca\(^{2+}\) conductance only slightly. The aspartate to histidine and cysteine mutations of Chronos and PsChR also influenced their photocurrent, ion permeability, kinetics, and light sensitivity. Most interestingly, PsChR D139H showed a much-improved photocurrent, compared to wild type, and even higher Na+ selectivity to H\(^+\) than XXM. PsChR D139H also showed a strongly enhanced Ca\(^{2+}\) conductance, more than two-fold that of the CatCh. (4) Conclusions: We found that mutating the aspartate of the TM4 influences the ion selectivity of channelrhodopsins. With the large photocurrent and enhanced Na\(^+\) selectivity and Ca\(^{2+}\) conductance, XXM and PsChR D139H are promising powerful optogenetic tools, especially for Ca\(^{2+}\) manipulation.
The green synthesis of silver nanoparticles (SNPs) using plant extracts is an eco-friendly method. It is a single step and offers several advantages such as time reducing, cost-effective and environmental non-toxic. Silver nanoparticles are a type of Noble metal nanoparticles and it has tremendous applications in the field of diagnostics, therapeutics, antimicrobial activity, anticancer and neurodegenerative diseases. In the present work, the aqueous extracts of aerial parts of Lampranthus coccineus and Malephora lutea F. Aizoaceae were successfully used for the synthesis of silver nanoparticles. The formation of silver nanoparticles was early detected by a color change from pale yellow to reddish-brown color and was further confirmed by transmission electron microscope (TEM), UV–visible spectroscopy, Fourier transform infrared (FTIR) spectroscopy, dynamic light scattering (DLS), X-ray diffraction (XRD), and energy-dispersive X-ray diffraction (EDX). The TEM analysis of showed spherical nanoparticles with a mean size between 12.86 nm and 28.19 nm and the UV- visible spectroscopy showed λ\(_{max}\) of 417 nm, which confirms the presence of nanoparticles. The neuroprotective potential of SNPs was evaluated by assessing the antioxidant and cholinesterase inhibitory activity. Metabolomic profiling was performed on methanolic extracts of L. coccineus and M. lutea and resulted in the identification of 12 compounds, then docking was performed to investigate the possible interaction between the identified compounds and human acetylcholinesterase, butyrylcholinesterase, and glutathione transferase receptor, which are associated with the progress of Alzheimer’s disease. Overall our SNPs highlighted its promising potential in terms of anticholinesterase and antioxidant activity as plant-based anti-Alzheimer drug and against oxidative stress.
In plants, antimicrobial immune responses involve the cellular release of anions and are responsible for the closure of stomatal pores. Detection of microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) induces currents mediated via slow-type (S-type) anion channels by a yet not understood mechanism. Here, we show that stomatal closure to fungal chitin is conferred by the major PRRs for chitin recognition, LYK5 and CERK1, the receptor-like cytoplasmic kinase PBL27, and the SLAH3 anion channel. PBL27 has the capacity to phosphorylate SLAH3, of which S127 and S189 are required to activate SLAH3. Full activation of the channel entails CERK1, depending on PBL27. Importantly, both S127 and S189 residues of SLAH3 are required for chitin-induced stomatal closure and anti-fungal immunity at the whole leaf level. Our results demonstrate a short signal transduction module from MAMP recognition to anion channel activation, and independent of ABA-induced SLAH3 activation.
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.