Internal Structure of Nanometer-Sized Droplets Prepared by Antisolvent Precipitation.

Antisolvent precipitation (AP) is a low-cost and less-invasive preparation alternative for organic nanoparticles compared to top-down methods such as high-pressure homogenization or milling. Here we report on particularly small organic nanoparticles (NPs) prepared by AP. It has been found for various materials that these NPs in their liquid state exhibit a significant degree of molecular order at their interface toward the dispersion medium including ubiquinones (coenzyme Q10), triglycerides (trimyristin, tripalmitin), and alkanes (tetracosane). This finding is independent of the use of a stabilizer in the formulation. While this is obviously a quite general interfacial structuring effect, the respective structural details of specific NPs systems might differ. Here, a detailed structural characterization of very small liquid coenzyme Q10 (Q10) NPs is presented as a particular example for this phenomenon. The Q10 NPs have been prepared by AP in the presence of two different stabilizers, sodium dodecyl sulfate (SDS) and pentaethylene glycol monododecyl ether (C12E5), respectively, and without any stabilizer. The NPs' size is initially analyzed by photon correlation spectroscopy (PCS). The SDS-stabilized Q10 NPs have been studied further by differential scanning calorimetry (DSC), small-angle X-ray and neutron scattering (SAXS, SANS), wide-angle X-ray scattering (WAXS), and cryogenic transmission electron microscopy (CryoTEM). A simultaneous analysis of SAXS and contrast variation SANS studies revealed the molecular arrangement within the interface between the NPs and the dispersion medium. The Q10 NPs stabilized by SDS and C12E5, respectively, are small (down to 19.9 nm) and stable (for at least 16 months) even when no stabilizer is used. The SDS-stabilized Q10 NPs reported here, are therewith, to the best of our knowledge, the smallest organic NPs which have been reported to be prepared by AP so far. In particular, these NPs exhibit a core-shell structure consisting of an amorphous Q10 core and a surrounding shell, which is mainly composed of oriented Q10 molecules and aligned SDS molecules. This structure suggests a significant amphiphilic behavior and a rather unexpected stabilizing role of Q10 molecules.

Climate change and spring frost damages for sweet cherries in Germany.

Spring frost can be a limiting factor in sweet cherry (Prunus avium L.) production. Rising temperatures in spring force the development of buds, whereby their vulnerability to freezing temperatures continuously increases. With the beginning of blossom, flowers can resist only light frosts without any significant damage. In this study, we investigated the risk of spring frost damages during cherry blossom for historical and future climate conditions at two different sites in NE (Berlin) and SW Germany (Geisenheim). Two phenological models, developed on the basis of phenological observations at the experimental sweet cherry orchard in Berlin-Dahlem and validated for endodormancy release and for warmer climate conditions (already published), were used to calculate the beginning of cherry blossom in Geisenheim, 1951-2015 (external model validation). Afterwards, on the basis of a statistical regionalisation model WETTREG (RCP 8.5), the frequency of frost during cherry blossom was calculated at both sites for historical (1971-2000) and future climate conditions (2011-2100). From these data, we derived the final flower damage, defined as the percentage of frozen flowers due to single or multiple frost events during blossom. The results showed that rising temperatures in this century can premature the beginning of cherry blossom up to 17 days at both sites, independent of the used phenological model. The frequency and strength of frost was characterised by a high temporal and local variability. For both sites, no significant increase in frost frequency and frost damage during blossom was found. In Geisenheim, frost damages significantly decreased from the middle of the twenty-first century. This study additionally emphasises the importance of reliable phenological models which not only work for current but also for changed climate conditions and at different sites. The date of endodormancy release should always be a known parameter in chilling/forcing models.

Selected Plant Metabolites Involved in Oxidation-Reduction Processes during Bud Dormancy and Ontogenetic Development in Sweet Cherry Buds (Prunus avium L.).

Many biochemical processes are involved in regulating the consecutive transition of different phases of dormancy in sweet cherry buds. An evaluation based on a metabolic approach has, as yet, only been partly addressed. The aim of this work, therefore, was to determine which plant metabolites could serve as biomarkers for the different transitions in sweet cherry buds. The focus here was on those metabolites involved in oxidation-reduction processes during bud dormancy, as determined by targeted and untargeted mass spectrometry-based methods. The metabolites addressed included phenolic compounds, ascorbate/dehydroascorbate, reducing sugars, carotenoids and chlorophylls. The results demonstrate that the content of phenolic compounds decrease until the end of endodormancy. After a long period of constancy until the end of ecodormancy, a final phase of further decrease followed up to the phenophase open cluster. The main phenolic compounds were caffeoylquinic acids, coumaroylquinic acids and catechins, as well as quercetin and kaempferol derivatives. The data also support the protective role of ascorbate and glutathione in the para- and endodormancy phases. Consistent trends in the content of reducing sugars can be elucidated for the different phenophases of dormancy, too. The untargeted approach with principle component analysis (PCA) clearly differentiates the different timings of dormancy giving further valuable information.

Spring frost vulnerability of sweet cherries under controlled conditions.

Spring frost is a significant production hazard in nearly all temperate fruit-growing regions. Sweet cherries are among the first fruit varieties starting their development in spring and therefore highly susceptible to late frost. Temperatures at which injuries are likely to occur are widely published, but their origin and determination methods are not well documented. In this study, a standardized method was used to investigate critical frost temperatures for the sweet cherry cultivar 'Summit' under controlled conditions. Twigs were sampled at four development stages ("side green," "green tip," "open cluster," "full bloom") and subjected to three frost temperatures (-2.5, -5.0, -10.0 °C). The main advantage of this method, compared to other approaches, was that the exposition period and the time interval required to reach the target temperature were always constant (2 h). Furthermore, then, the twigs were placed in a climate chamber until full bloom, before the examination of the flowers and not further developed buds started. For the first two sampling stages (side green, green tip), the number of buds found in open cluster, "first white," and full bloom at the evaluation date decreased with the strength of the frost treatment. The flower organs showed different levels of cold hardiness and became more vulnerable in more advanced development stages. In this paper, we developed four empirical functions which allow calculating possible frost damages on sweet cherry buds or flowers at the investigated development stages. These equations can help farmers to estimate possible frost damages on cherry buds due to frost events. However, it is necessary to validate the critical temperatures obtained in laboratory with some field observations.

Mesoscopic Structure of Lipid Nanoparticle Formulations for mRNA Drug Delivery: Comirnaty and Drug-Free Dispersions.

Lipid nanoparticles (LNPs) produced by antisolvent precipitation (ASP) are used in formulations for mRNA drug delivery. The mesoscopic structure of such complex multicomponent and polydisperse nanoparticulate systems is most relevant for their drug delivery properties, medical efficiency, shelf life, and possible side effects. However, the knowledge on the structural details of such formulations is very limited. Essentially no such information is publicly available for pharmaceutical dispersions approved by numerous medicine agencies for the use in humans and loaded with mRNA encoding a mimic of the spike protein of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) as, e.g., the Comirnaty formulation (BioNTech/Pfizer). Here, we present a simple preparation method to mimic the Comirnaty drug-free LNPs including a comparison of their structural properties with those of Comirnaty. Strong evidence for the liquid state of the LNPs in both systems is found in contrast to the designation of the LNPs as solid lipid nanoparticles by BioNTech. An exceptionally detailed and reliable structural model for the LNPs i.a. revealing their unexpected narrow size distribution will be presented based on a combined small-angle X-ray scattering and photon correlation spectroscopy (SAXS/PCS) evaluation method. The results from this experimental approach are supported by light microscopy, 1H NMR spectroscopy, Raman spectroscopy, cryogenic electron microscopy (cryoTEM), and simultaneous SAXS/SANS studies. The presented results do not provide direct insights on particle formation or dispersion stability but should contribute significantly to better understanding the LNP drug delivery process, enhancing their medical benefit, and reducing side effects.

Barley grains, deficient in cytosolic small subunit of ADP-glucose pyrophosphorylase, reveal coordinate adjustment of C:N metabolism mediated by an overlapping metabolic-hormonal control.

The barley Risø16 mutation leads to inactivation of cytosolic ADP-Glc pyrophosphorylase, and results in decreased ADP-Glc and endospermal starch levels. Here we show that this mutation is accompanied by a decrease in storage protein accumulation and seed size, which indicates that alteration of a single enzymatic step can change the network of storage metabolism as a whole. We used comprehensive transcript, metabolite and hormonal profiling to compare grain metabolism and development of Risø16 and wild-type endosperm. Despite increased sugar availability in mutant endosperm, glycolytic intermediates downstream of hexose phosphates remained unchanged or decreased, while several glycolytic enzymes were downregulated at the transcriptional level. Metabolite and transcript profiling also indicated an inhibition of the tricarboxylic acid cycle at the level of mitochondrial nicotinamide adenine dinucleotide (NAD)-isocitrate dehydrogenase and an attendant decrease in alpha-ketoglutarate and amino acids levels in Risø16, compared with wild type. Decreased levels of cytokinins in Risø16 endosperm suggested co-regulation between starch synthesis, abscisic acid (ABA) deficiency and cytokinin biosynthesis. Comparative cis-element analysis in promoters of jointly downregulated genes in Risø16 revealed an overlap between metabolic and hormonal regulation, which leds to a coordinated downregulation of endosperm-specific and ABA-inducible gene expression (storage proteins) together with repression by sugars (isocitrate dehydrogenase, amylases). Such co-regulation ensured that decreased carbon fluxes into starch lead to a coordinated inhibition of glycolysis, amino acid and storage proteins biosynthesis, which is useful in the prevention of osmotic imbalances and oxidative stress due to increased accumulation of sugars.

Metabolites That Confirm Induction and Release of Dormancy Phases in Sweet Cherry Buds.

Here we report on metabolites found in a targeted profiling of 'Summit' flower buds for nine years, which could be indicators for the timing of endodormancy release (t1) and beginning of ontogenetic development (t1*). Investigated metabolites included chrysin, arabonic acid, pentose acid, sucrose, abscisic acid (ABA), and abscisic acid glucose ester (ABA-GE). Chrysin and water content showed an almost parallel course between leaf fall and t1*. After 'swollen bud', water content raised from ~60 to ~80% at open cluster, while chrysin content decreased and lost its function as an acetylcholinesterase inhibitor. Both parameters can be suitable indicators for t1*. Arabonic acid showed a clear increase after t1*. Pentose acid would be a suitable metabolite to identify t1 and t1*, but would not allow describing the ecodormancy phase, because of its continuously low value during this time. Sucrose reached a maximum during ecodormancy and showed a significant correlation with air temperature, which confirms its cryoprotective role in this phase. The ABA content showed maximum values during endodormancy and decreased during ecodormancy, reaching 50% of its content t1 at t1*. It appears to be the key metabolite to define the ecodormancy phase. The ABA-GE was present at all stages and phases and was much higher than the ABA content and is a readily available storage pool in cherry buds.

Metabolites in Cherry Buds to Detect Winter Dormancy.

Winter dormancy is still a "black box" in phenological models, because it evades simple observation. This study presents the first step in the identification of suitable metabolites which could indicate the timing and length of dormancy phases for the sweet cherry cultivar 'Summit'. Global metabolite profiling detected 445 named metabolites in flower buds, which can be assigned to different substance groups such as amino acids, carbohydrates, phytohormones, lipids, nucleotides, peptides and some secondary metabolites. During the phases of endo- and ecodormancy, the energy metabolism in the form of glycolysis and the tricarboxylic acid (TCA) cycle was shut down to a minimum. However, the beginning of ontogenetic development was closely related to the up-regulation of the carbohydrate metabolism and thus to the generation of energy for the growth and development of the sweet cherry buds. From the 445 metabolites found in cherry buds, seven were selected which could be suitable markers for the ecodormancy phase, whose duration is limited by the date of endodormancy release (t1) and the beginning of ontogenetic development (t1*). With the exception of abscisic acid (ABA), which has been proven to control bud dormancy, all of these metabolites show nearly constant intensity during this phase.

ABA and Not Chilling Reduces Heat Requirement to Force Cherry Blossom after Endodormancy Release.

Models used to predict the onset of fruit tree blossom under changed climate conditions should be physiologically based as much as possible. Pure optimized phenology models carry the risk of unrealistic predictions due to a misinterpretation of metabolic processes. This was the motivation determining the relevant phases for chill and heat accumulation, which induces cherry blossom (cv. Summit). Investigations are based on 8 years of observational and analytical data, as well as on controlled experiments. For 'Summit' buds, to be released from endodormancy, 43 chill portions from 1 September are necessary. After endodormancy release (t1), on average on 30 November, no further chilling is required, because no correlation between chill accumulation during ecodormancy and the subsequent heat accumulation until 'Summit' blossom exist. The declining amount of heat, which induces cherry blossom after t1-shown in several forcing experiments-seems to be the result of the declining bud's abscisic acid (ABA) content, up to ~50% until the beginning of ontogenetic development. Shortly after t1, when the bud's ABA content is high, a huge amount of heat is necessary to induce cherry blossom under controlled conditions. Heat requirement reduces during ecodormancy along with the reduction in the ABA content. According to these findings, plant development during ecodormancy is suppressed by low temperatures in the orchard and a slowly declining bud's ABA content. These results should lead to a better consideration of the ecodormancy phase in phenology models.

A novel experimental approach for nanostructure analysis: simultaneous small-angle X-ray and neutron scattering.

Exploiting small-angle X-ray and neutron scattering (SAXS/SANS) on the same sample volume at the same time provides complementary nanoscale structural information in two different contrast situations. Unlike an independent experimental approach, the truly combined SAXS/SANS experimental approach ensures the exactness of the probed samples, particularly for in situ studies. Here, an advanced portable SAXS system that is dimensionally suitable for installation in the D22 zone of ILL is introduced. The SAXS apparatus is based on a Rigaku switchable copper/molybdenum microfocus rotating-anode X-ray generator and a DECTRIS detector with a changeable sample-to-detector distance of up to 1.6 m in a vacuum chamber. A case study is presented to demonstrate the uniqueness of the newly established method. Temporal structural rearrangements of both the organic stabilizing agent and organically capped gold colloidal particles during gold nanoparticle growth are simultaneously probed, enabling the immediate acquisition of correlated structural information. The new nano-analytical method will open the way for real-time investigations of a wide range of innovative nanomaterials and will enable comprehensive in situ studies on biological systems. The potential development of a fully automated SAXS/SANS system with a common control environment and additional sample environments, permitting a continual and efficient operation of the system by ILL users, is also introduced.

Effects of N-application on utilization of 15N and 13C and quality in two wheat cultivars.

We studied N uptake and distribution in wheat, and the incorporation of nitrogen and carbon into gluten and non-gluten proteins using a double-labelling approach with 15N and 13C. Doses of N-fertilizer were split and applied at emergence, onset of stem elongation, and heading at rates of 280/140/140 mg N pot(-1), respectively simulating 90/45/45 kg N ha(-1). Five different combinations of N-fertilizations containing no or 10 % 15N were performed. The recovery of 15N added at the stages emergence, stem elongation or heading were 42, 60, and 64 %. Application of 15N at all three stages yielded in 51 % recovery. Remobilisation of straw N was greater for Golia. The 15N concentration in gluten proteins of Golia show higher values than Gonen. The ratio of 15N gluten/15N non-gluten proteins of Golia were higher, which implies a lower non-gluten protein activity during grain filling. The 13C concentration in gluten and non-gluten proteins did not differ between both cultivars.

15N allocation into wheat grains (Triticum aestivum L.) influenced by sowing rate and water supply at flowering under a Mediterranean climate.

This study examined the effects of a reduced wheat sowing rate (250 vs. 500 grains m-2) on grain yield, uptake of 15N into grains, and the incorporation into gluten and non-gluten proteins of wheat under field conditions in the Aegean region. A single 15N application was applied at stem elongation, at flowering, or at both developmental stages. Each 15N treatment included either additional water supply, or no additional water supply at flowering. Sowing rate (either 250 or 500 grains m-2) had no impact on grain yield. Grain yield increased with additional water supply, but at the expense of protein quality, because of a decrease in the protein content of gluten. The 15N content of the gluten and non-gluten proteins at grain maturity was not different among cultivars. 15N applied at both stem elongation and flowering was found in comparable amounts in grains and protein fractions, irrespective of sowing rate.

Ectopic expression of phosphoenolpyruvate carboxylase in Vicia narbonensis seeds: effects of improved nutrient status on seed maturation and transcriptional regulatory networks.

Seed maturation responds to endogenous and exogenous signals like nutrient status, energy and hormones. We recently showed that phosphoenolpyruvate carboxylase (PEPC) overexpression in Vicia narbonensis seeds alters seed metabolism and channels carbon into organic acids, resulting in greater seed storage capacity and increased protein content. Thus, these lines represent models with altered sink strength and improved nutrient status. Here we analyse seed developmental and metabolic parameters, and C/N partitioning in these seeds. Transgenic embryos take up more carbon and nitrogen. Changes in dry to FW ratio, seed fill duration and major seed components indicate altered seed development. Array-based gene expression analysis of embryos reveals upregulation of seed metabolism, especially during the transition phase and at late maturation, in terms of protein storage and processing, amino acid metabolism, primary metabolism and transport, energy and mitochondrial activity, transcriptional and translational activity, stress tolerance, photosynthesis, cell proliferation and elongation, signalling and hormone action and regulated protein degradation. Stimulated cell elongation is in accordance with upregulated signalling pathways related to gibberellic acid/brassinosteroids. We discuss that activated organic and amino acid production leads to a wide-range activation of nitrogen metabolism, including the machinery of storage protein synthesis, amino acid synthesis, protein processing and deposition, translational activity and the methylation cycle. We suggest that alpha-ketoglutarate (alpha-KG) and/or oxalacetate provide signals for coordinate upregulation of amino acid biosynthesis. Activation of stress tolerance genes indicates partial overlap between nutrient, stress and abscisic acid (ABA) signals, indicating a common interacting or regulatory mechanism between nutrients, stress and ABA. In conclusion, analysis of PEPC overexpressing seeds identified pathways responsive to metabolic and nutrient control on the transcriptional level and its underlying signalling mechanisms.

Uptake and allocation of carbon and nitrogen in Vicia narbonensis plants with increased seed sink strength achieved by seed-specific expression of an amino acid permease.

Over-expressing an amino acid permease in Vicia narbonensis seeds increases sink strength for N that is evident from the higher seed protein content and seed weight. Here, the effect of increased seed sink strength of line AAP-12 on growth, development, and on whole plant carbon and nitrogen uptake and partitioning is analysed. AAP-12 plants have a prolonged growth period. Accumulation and partitioning of dry matter and N in leaves, stems, and pods are higher whereas remobilization to the seeds is delayed, indicating that the switch from growth to reserve allocation and remobilization is delayed. Measuring uptake and allocation of (15)N-ammonia applied via the roots revealed a higher and longer label uptake period during maturation. Measuring whole plant carbon fixation and allocation after (13)C labelling shows higher levels at maturation, particularly in seeds, indicating higher seed sink strength for C and increased allocation into maturing seeds. Levels of cytokinins were dramatically increased in AAP-12 seeds indicating its role in nitrogen-mediated growth stimulation. AAP-12 seeds have higher natural abundances for (13)C indicating increased C fixation via PEP carboxylase in order to meet the higher demand of carbon acceptors for amino acid synthesis. In summary, increased seed sink strength for N in AAP-12 stimulates seed growth, but also that of vegetative organs, which finally leads to a higher ratio of vegetative to seed biomass at maturity and thus a lower harvest index. Therefore, the increased N uptake due to higher seed demand of AAP-12 is partly compensated by growth stimulation of vegetative organs.

Ectopic expression of an amino acid transporter (VfAAP1) in seeds of Vicia narbonensis and pea increases storage proteins.

Storage protein synthesis is dependent on available nitrogen in the seed, which may be controlled by amino acid import via specific transporters. To analyze their rate-limiting role for seed protein synthesis, a Vicia faba amino acid permease, VfAAP1, has been ectopically expressed in pea (Pisum sativum) and Vicia narbonensis seeds under the control of the legumin B4 promoter. In mature seeds, starch is unchanged but total nitrogen is 10% to 25% higher, which affects mainly globulin, vicilin, and legumin, rather than albumin synthesis. Transgenic seeds in vitro take up more [14C]-glutamine, indicating increased sink strength for amino acids. In addition, more [14C] is partitioned into proteins. Levels of total free amino acids in growing seeds are unchanged but with a shift toward higher relative abundance of asparagine, aspartate, glutamine, and glutamate. Hexoses are decreased, whereas metabolites of glycolysis and the tricarboxylic acid cycle are unchanged or slightly lower. Phosphoenolpyruvate carboxylase activity and the phosphoenolpyruvate carboxylase-to-pyruvate kinase ratios are higher in seeds of one and three lines, indicating increased anaplerotic fluxes. Increases of individual seed size by 20% to 30% and of vegetative biomass indicate growth responses probably due to improved nitrogen status. However, seed yield per plant was not altered. Root application of [15N] ammonia results in significantly higher label in transgenic seeds, as well as in stems and pods, and indicates stimulation of nitrogen root uptake. In summary, VfAAP1 expression increases seed sink strength for nitrogen, improves plant nitrogen status, and leads to higher seed protein. We conclude that seed protein synthesis is nitrogen limited and that seed uptake activity for nitrogen is rate limiting for storage protein synthesis.