In plant cells, plastids form elongated extensions called stromules, the regulation and purposes of which remain unclear. Here, we quantitatively explore how different stromule structures serve to enhance the ability of a plastid to interact with other organelles: increasing the effective space for interaction and biomolecular exchange between organelles. Interestingly, electron microscopy and confocal imaging showed that the cytoplasm in Arabidopsis thaliana and Nicotiana benthamiana epidermal cells is extremely thin (around 100 nm in regions without organelles), meaning that inter-organelle interactions effectively take place in 2D. We combine these imaging modalities with mathematical modeling and new in planta experiments to demonstrate how different stromule varieties (single or multiple, linear or branching) could be employed to optimize different aspects of inter-organelle interaction capacity in this 2D space. We found that stromule formation and branching provide a proportionally higher benefit to interaction capacity in 2D than in 3D. Additionally, this benefit depends on optimal plastid spacing. We hypothesize that cells can promote the formation of different stromule architectures in the quasi-2D cytoplasm to optimize their interaction interface to meet specific requirements. These results provide new insight into the mechanisms underlying the transition from low to high stromule numbers, the consequences for interaction with smaller organelles, how plastid access and plastid to nucleus signaling are balanced and the impact of plastid density on organelle interaction.

Novel unimolecular bivalent glycoconjugates were assembled combining several functionalized capsular polysaccharides of Streptococcus pneumoniae and Neisseria meningitidis to a carrier protein by using an effective strategy based on the Ugi 4-component reaction. The development of multivalent glycoconjugates opens new opportunities in the field of vaccine design, but their high structural complexity involves new analytical challenges. Nuclear Magnetic Resonance has found wide applications in the characterization and impurity profiling of carbohydrate-based vaccines. Eight bivalent conjugates were studied by quantitative NMR analyzing the structural identity, the content of each capsular polysaccharide, the ratios between polysaccharides, the polysaccharide to protein ratios and undesirable contaminants. The qNMR technique involves experiments with several modified parameters for obtaining spectra with quantifiable signals. In addition, the achieved NMR results were combined with the results of colorimetric assay and Size Exclusion HPLC for assessing the protein content and free protein percentage, respectively. The application of quantitative NMR showed to be efficient to clear up the new structural complexities while allowing the quantitative assessment of the components.

Ceramides (CERs) play a major role in skin barrier function and direct replacement of depleted skin CERs,due to skin disorder or aging, has beneficial effects in improving skin barrier function and skin hydration.Though, plants are reliable source of CERs, absence of economical and effective method of hydrolysis toconvert the dominant plant sphingolipid, glucosylceramides (GlcCERs), into CERs remains a challenge.This study aims at exploring alternative GlcCERs sources and chemical method of hydrolysis into CERsfor dermal application. GlcCERs isolated from lupin bean (Lupinus albus), mung bean (Vigna radiate) andnaked barley (Hordium vulgare) were identified using ultra high performance liquid chromatographyhyphenated with atmospheric pressure chemical ionization - high resolution tandem mass spectrometer(UHPLC/APCI-HRMS/MS) and quantified with validated automated multiple development-high perfor-mance thin layer chromatography (AMD-HPTLC) method. Plant GlcCERs were hydrolyzed into CERs withmild acid hydrolysis (0.1 N HCl) after treating them with oxidizing agent, NaIO4,and reducing agent,NaBH4. GlcCERs with 4,8-sphingadienine, 8-sphingenine and 4-hydroxy-8-sphingenine sphingoid baseslinked with C14 to C26 -hydroxylated fatty acids (FAs) were identified. Single GlcCER (m/z 714.5520)was dominant in lupin and mung beans while five major GlcCERs species (m/z 714.5520, m/z 742.5829,m/z 770.6144, m/z 842.6719 and m/z 844.56875) were obtained from naked barley. The GlcCERs con-tents of the three plants were comparable. However, lupin bean contains predominantly (> 98 %) a singleGlcCER (m/z 714.5520). Considering the affordability, GlcCER content and yield, lupin bean would bethe preferred alternative commercial source of GlcCERs. CER species bearing 4,8-sphingadienine and 8-sphingenine sphingoid bases attached to C14 to 24 FAs were found after mild acid hydrolysis. CER specieswith m/z 552.4992 was the main component in the beans while CER with m/z 608.5613 was dominantin the naked barley. However, CERs with 4-hydroxy-8-sphingenine sphingoid base were not detected inUHPLC-HRMS/MS study suggesting that the method works for mainly GlcCERs carrying dihydroxy sph-ingoid bases. The method is economical and effective which potentiates the commercialization of plantCERs for dermal application.

Salvadora persica L. (toothbrush tree, Miswak) is well recognized in most Middle Eastern and African countries for its potential role in dental care, albeit the underlying mechanism for its effectiveness is still not fully understood. A comparative MS and NMR metabolomics approach was employed to investigate the major primary and secondary metabolites composition of S. persica in context of its organ type viz., root or stem to rationalize for its use as a tooth brush. NMR metabolomics revealed its enrichment in nitrogenous compounds including proline-betaines i.e., 4-hydroxy-stachydrine and stachydrine reported for the first time in S. persica. LC/MS metabolomics identified flavonoids (8), benzylurea derivatives (5), butanediamides (3), phenolic acids (8) and 5 sulfur compounds, with 21 constituents reported for the first time in S. persica. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) of either NMR or LC/MS dataset clearly separated stem from root specimens based on nitrogenous compounds abundance in roots and is justifying for its preference as toothbrush versus stems. The presence of betaines at high levels in S. persica (9−12 μg/mg dry weight) offers novel insights into its functioning as an osmoprotectant that maintains the hydration of oral mucosa. Additionally, the previously described anti-inflammatory activity of stachydrine along with the antimicrobial effects of sulfonated flavonoids, benzylisothiocynate and ellagic acid derivatives are likely contributors to S. persica oral hygiene health benefits. Among root samples, variation in sugars and organic acids levels were the main discriminatory criterion. This study provides the first standardization of S. persica extract using qNMR for further inclusion in nutraceuticals.

Plant cell walls are sophisticated carbohydrate-rich structures representing the immediate contact surface with the extracellular environment, often serving as the first barrier against biotic and abiotic stresses. Notably, a variety of perturbations in plant cell walls result in upregulated jasmonate (JA) production, a phytohormone with essential roles in defense and growth responses. Hence, cell wall-derived signals can initiate intracellular JA-mediated responses and the elucidation of the underlying signaling pathways could provide novel insights into cell wall maintenance and remodeling, as well as advance our understanding on how is JA biosynthesis initiated. This Mini Review will describe current knowledge about cell wall-derived damage signals and their effects on JA biosynthesis, as well as provide future perspectives.

Knipholone (1) and knipholone anthrone (2), isolated from the Ethiopian medicinal plant Kniphofia foliosa Hochst. are two phenyl anthraquinone derivatives, a compound class known for biological activity. In the present study, we describe the activity of both 1 and 2 in several biological assays including cytotoxicity against four human cell lines (Jurkat, HEK293, SH-SY5Y and HT-29), antiplasmodial activity against Plasmodium falciparum 3D7 strain, anthelmintic activity against the model organism Caenorhabditis elegans, antibacterial activity against Aliivibrio fischeri and Mycobacterium tuberculosis and anti-HIV-1 activity in peripheral blood mononuclear cells (PBMCs) infected with HIV-1c. In parallel, we investigated the stability of knipholone (2) in solution and in culture media. Compound 1 displays strong cytotoxicity against Jurkat, HEK293 and SH-SY5Y cells with growth inhibition ranging from approximately 62–95% when added to cells at 50 μM, whereas KA (2) exhibits weak to strong activity with 26, 48 and 70% inhibition of cell growth, respectively. Both 1 and 2 possess significant antiplasmodial activity against Plasmodium falciparum 3D7 strain with IC50 values of 1.9 and 0.7 μM, respectively. These results complement previously reported data on the cytotoxicity and antiplasmodial activity of 1 and 2. Furthermore, compound 2 showed HIV-1c replication inhibition (growth inhibition higher than 60% at tested concentrations 0.5, 5, 15 and 50 μg/ml and an EC50 value of 4.3 μM) associated with cytotoxicity against uninfected PBMCs. The stability study based on preincubation, HPLC and APCI-MS (atmospheric-pressure chemical ionization mass spectrometry) analysis indicates that compound 2 is unstable in culture media and readily oxidizes to form compound 1. Therefore, the biological activity attributed to 2 might be influenced by its degradation products in media including 1 and other possible dimers. Hence, bioactivity results previously reported from this compound should be taken with caution and checked if they differ from those of its degradation products. To the best of our knowledge, this is the first report on the anti-HIV activity and stability analysis of compound 2.

Trigonelline (3-carboxy-1-methyl pyridinium) was identified as a relevant bioactivity and taste imparting component in Balanites aegyptiaca fruit, using 1H NMR of crude extracts without any fractionation or isolation step. The structural integrity of trigonelline was established within the extract matrix via1H NMR, 1H–1H COSY, HMQC and HMBC and by comparison with authentic standard. A quantitative 1H NMR method (qHNMR) was used to determine trigonelline concentrations in the peel and pulp of B. aegyptiaca fruit of 8 and 13 mg g−1, respectively. Trigonelline so far has not been reported from B. aegyptiaca or its genus as it easily escapes LC–MS based detection. Its discovery provides novel insight into the balanite fruits antidiabetic properties as the compound is known for a pronounced hypoglycemic effect. In addition, it is likely to impart the perceptible bitter taste portion to balanites sweet bitter taste. UPLC–MS of the crude extract additionally revealed the fruit flavonoid pattern showing quercetin/isorhamnetin flavonol conjugates in addition to epicatechin, the latter being present at much lower levels.

Recognition of pathogen-associated molecular patterns (PAMPs) induces multiple defense mechanisms to limit pathogen growth. Here, we show that the Arabidopsis thaliana tandem zinc finger protein 9 (TZF9) is phosphorylated by PAMP-responsive mitogen-activated protein kinases (MAPKs) and is required to trigger a full PAMP-triggered immune response. Analysis of a tzf9 mutant revealed attenuation in specific PAMP-triggered reactions such as reactive oxygen species accumulation, MAPK activation and, partially, the expression of several PAMP-responsive genes. In accordance with these weaker PAMP-triggered responses, tzf9 mutant plants exhibit enhanced susceptibility to virulent Pseudomonas syringae pv. tomato DC3000. Visualization of TZF9 localization by fusion to green fluorescent protein revealed cytoplasmic foci that co-localize with marker proteins of processing bodies (P-bodies). This localization pattern is affected by inhibitor treatments that limit mRNA availability (such as cycloheximide or actinomycin D) or block nuclear export (leptomycin B). Coupled with its ability to bind the ribohomopolymers poly(rU) and poly(rG), these results suggest involvement of TZF9 in post-transcriptional regulation, such as mRNA processing or storage pathways, to regulate plant innate immunity.

Nicotiana tabacum (tobacco) plants have short and long glandular trichomes. There is evidence that tobacco trichomes play several roles in the defense against biotic and abiotic stresses. cDNA libraries were constructed from control and cadmium (Cd)-treated leaf trichomes. Almost 2,000 expressed sequence tag (EST) cDNA clones were sequenced to analyze gene expression in control and Cd-treated leaf trichomes. Genes for stress response as well as for primary metabolism scored highly, indicating that the trichome is a biologically active and stress-responsive tissue. Reverse transcription–PCR (RT–PCR) analysis demonstrated that antipathogenic T-phylloplanin-like proteins, glutathione peroxidase and several classes of pathogenesis-related (PR) proteins were expressed specifically or dominantly in trichomes. Cysteine-rich PR proteins, such as non-specific lipid transfer proteins (nsLTPs) and metallocarboxypeptidase inhibitors, are candidates for the sequestration of metals. The expression of osmotin and thaumatin-like proteins was induced by Cd treatment in both leaves and trichomes. Confocal laser scanning microscopy (CLSM) showed that glutathione levels in tip cells of both long and short trichomes were higher than those in other types of leaf cells, indicating the presence of an active sulfur-dependent protective system in trichomes. Our results revealed that the trichome-specific transcriptome approach is a powerful tool to investigate the defensive functions of trichomes against both abiotic and biotic stress. Trichomes are shown to be an enriched source of useful genes for molecular breeding towards stress-tolerant plants.

HPLC-UV and MS/MS studies of impurity profiles of original (Xenical®, F. Hoffmann-La Roche Ltd., Switzerland) and generic (Cobese™, Ranbaxy Laboratories Limited, India, and Orsoten, KRKA, Russia) products were carried out. The drug and related impurities were extracted by dissolving commercial samples in ethanol. The generic formulations contained higher levels of impurities than the original product. Impurity profiles (HPLC-MS/MS) of the generic samples are similar among themselves, whilst different in comparison to the impurity profile of the original product. The number of detected impurities for generics (14 impurities in Cobese™ and 13 impurities in Orsoten) is higher than for the original product (3 impurities in Xenical®). Based on these analyses the overall analytical quality follows the order Xenical® (best) > Orsoten > Cobese™.