Practical guide toward discovery of biomolecules with biostimulant activity.

The growing demand for sustainable solutions in agriculture, critical for crop productivity and food quality in the face of climate change and reduced agrochemical usage, has brought biostimulants into the spotlight as valuable tools for regenerative agriculture. Due to their diverse biological activities, biostimulants contribute to crop growth, nutrient use efficiency, abiotic resilience, and soil health restoration. Biomolecules, including but not limited to humic substances, protein lysates, phenolics and carbohydrates have undergone thorough investigation because of their demonstrated biostimulant activities. Here, we review the process of discovery and development of extract-based biostimulants and propose a practical step-by-step pipeline starting with biomolecule investigation, followed by extraction and isolation, bioactivity determination, identification of active compound(s), mechanistic elucidation, formulation, and effectiveness assessment. The different steps generate a roadmap that aims to expedite the transfer of interdisciplinary knowledge from laboratory-scale studies to pilot-scale production in practical scenarios aligned with the regulatory framework.

Synthesis of Mixed Phosphonate Esters and Amino Acid-Based Phosphonamidates, and Their Screening as Herbicides.

While organophosphorus chemistry is gaining attention in a variety of fields, the synthesis of the phosphorus derivatives of amino acids remains a challenging task. Previously reported methods require the deprotonation of the nucleophile, complex reagents or hydrolysis of the phosphonate ester. In this paper, we demonstrate how to avoid these issues by employing phosphonylaminium salts for the synthesis of novel mixed n-alkylphosphonate diesters or amino acid-derived n-alkylphosphonamidates. We successfully applied this methodology for the synthesis of novel N-acyl homoserine lactone analogues with varying alkyl chains and ester groups in the phosphorus moiety. Finally, we developed a rapid, quantitative and high-throughput bioassay to screen a selection of these compounds for their herbicidal activity. Together, these results will aid future research in phosphorus chemistry, agrochemistry and the synthesis of bioactive targets.

Altering the Sex Pheromone Cyclo(l-Pro-l-Pro) of the Diatom Seminavis robusta towards a Chemical Probe.

As a major group of algae, diatoms are responsible for a substantial part of the primary production on the planet. Pennate diatoms have a predominantly benthic lifestyle and are the most species-rich diatom group, with members of the raphid clades being motile and generally having heterothallic sexual reproduction. It was recently shown that the model species Seminavis robusta uses multiple sexual cues during mating, including cyclo(l-Pro-l-Pro) as an attraction pheromone. Elaboration of the pheromone-detection system is a key aspect in elucidating pennate diatom life-cycle regulation that could yield novel fundamental insights into diatom speciation. This study reports the synthesis and bio-evaluation of seven novel pheromone analogs containing small structural alterations to the cyclo(l-Pro-l-Pro) pheromone. Toxicity, attraction, and interference assays were applied to assess their potential activity as a pheromone. Most of our analogs show a moderate-to-good bioactivity and low-to-no phytotoxicity. The pheromone activity of azide- and diazirine-containing analogs was unaffected and induced a similar mating behavior as the natural pheromone. These results demonstrate that the introduction of confined structural modifications can be used to develop a chemical probe based on the diazirine- and/or azide-containing analogs to study the pheromone-detection system of S. robusta.

The Use of PTI-Marker Genes to Identify Novel Compounds that Establish Induced Resistance in Rice.

Compounds that establish induced resistance (IR) in plants are promising alternatives for the pesticides that are progressively being banned worldwide. Screening platforms to identify IR-establishing compounds have been developed, but none were specifically designed for monocot plants. Here, we propose the use of an RT-qPCR screening platform, based on conserved immunity marker genes of rice as proxy for IR induction. Central regulators of biotic stress responses of rice were identified with a weighted gene co-expression network analysis (WGCNA), using more than 350 microarray datasets of rice under various sorts of biotic stress. Candidate genes were narrowed down to six immunity marker genes, based on consistent association with pattern-triggered immunity (PTI), both in rice plants as in rice cell suspension cultures (RCSCs). By monitoring the expression of these genes in RCSCs upon treatment with candidate IR-inducing compounds, we showed that our marker genes can predict IR induction in rice. Diproline, a novel IR-establishing compound for monocots that was detected with these marker genes, was shown to induce rice resistance against root-knot nematodes, without fitness costs. Gene expression profiling of the here-described PTI-marker genes can be executed on fully-grown plants or in RCSCs, providing a novel and versatile tool to predict IR induction.

Synthesis of 1,4-Oxazepane-2,5-diones via Cyclization of Rotationally Restricted Amino Acid Precursors and Structural Reassignment of Serratin.

Several natural products containing a 1,4-oxazepane-2,5-dione-core are known. One example is serratin, isolated from Serratia marcescens. Because of the presence of a carboxylic amide, which has a preference for a trans-conformation, and the presence of a labile lactone in this core, many synthetic methodologies commonly used for the cyclization toward medium-sized heterocycles cannot be applied. As N-acyl amino acids lacking a third substituent at nitrogen failed to undergo ring-closure, several N-protecting groups were evaluated. With the use of the removable PMB-group, an N-unsubstituted 1,4-oxazepane-2,5-dione was synthesized. Via the application of pseudoprolines (i.e. serine-derived oxazolidines as another type of protecting group), a compound with the presumed structure of the natural product serratin was obtained. As a result of the differences in spectral data, the incorrect structural assignment of the natural product serratin was identified. Instead of the predicted seven-membered heterocycle, a symmetrical serratamolide analogue is proposed to be the correct structure of serratin.

Detection and Toxicity Evaluation of Pyrrolizidine Alkaloids in Medicinal Plants Gynura bicolor and Gynura divaricata Collected from Different Chinese Locations.

Two edible plants in Southeast Asia, Gynura bicolor and G. divaricata, are not only known to be nutritive but also useful as medicinal herbs. Previous phytochemical investigation of Gynura species showed the presence of hepatotoxic pyrrolizidine alkaloids (PAs), indicating the toxic risk of using these two plants. The present study was designed to analyze the distribution of PA components and tried to evaluate the preliminary toxicity of these two Gynura species. Eight samples of G. bicolor and G. divaricata from five different Chinese locations were collected and their specific PAs were qualitatively characterized by applying an UPLC/MS/MS spectrometry method. Using a pre-column derivatization HPLC method, the total retronecine ester-type PAs in their alkaloids extracts were quantitatively estimated as well. Finally, their genotoxicity was investigated with an effective high-throughput screening method referred to as Vitotox™ test and their potential cytotoxicity was tested on HepG2 cells. It was found that different types of PAs were widely present in Gynura species collected from south of China. Among them, no significant genotoxic effects were detected with serial concentrations through the present in vitro assay. However, the cytotoxicity assay of Gynura plants collected from Jiangsu displayed weak activity at the concentration of 100 mg/ml. It is important to note that this research validates in part the indication that the use of Gynura species requires caution.

Synthesis of 2-Fluoro-1,4-benzoxazines and 2-Fluoro-1,4-benzoxazepin-5-ones by Exploring the Nucleophilic Vinylic Substitution (S(N)V) Reaction of gem-Difluoroenamides.

N-Benzoyl ß,ß-difluoroenamides and N-benzoyl fluoroynamides are novel structural units which have been explored as precursors in heterocyclic synthesis. The presence of two fluorine atoms at the ß-position of the enamide moiety endows unique electrophilic reactivity. Treatment of these enamides with oxygen nucleophiles gives rise to a nucleophilic vinylic substitution (S(N)V) reaction, which was directed toward 2-fluoro-1,4-benzoxazines and 2-fluoro-1,4-benzoxazepin-5-ones. Furthermore, fluorinated ynamides, a new type of building block, were prepared in excellent yields for the first time. In this case, ß-addition of nucleophiles across the triple bond is observed also.

Profiling and elucidation of the phenolic compounds in the aerial parts of Gynura bicolor and G. divaricata collected from different Chinese origins.

Gynura bicolor and G. divaricata are not only known to be nutritive as cultured vegetables, but also beneficial as folk medicines in East Asia. As demonstrated by the current phytochemical knowledge, the genus Gynura is a promising source of phenolics with multiple medicinal activities. To expand this phytochemical knowledge, the phenolic secondary metabolites of G. bicolor and G. divaricata were studied. From the aerial parts of these two species, collected in five different Chinese locations, two fractions of phenolic compounds with different polarity were obtained by extraction and chromatographic separation. Using UPLC/MS/MS analysis, a total of 53 phenolics were either identified by comparison with respective reference compounds or tentatively characterized by their chromatographic behavior, UV-absorption patterns, and MS fragmentations. Some naturally existing positional isomers of O-caffeoylquinic acid, O-p-coumaroylquinic acid, O-feruloylquinic acid, and dicaffeoylquinic acid as well as their methyl esters were qualitatively characterized by their specific fragmentation patterns in targeted MS/MS. In addition, the aerial parts of the two Gynura species contained kaempferol, quercetin oligoglycosides, and a variety of derivatives of benzoic acid, hydroxycinnamic acid, and caffeic acid. Furthermore, the distribution of phenolic compounds in the two species from different Chinese origins was discussed. Finally, an investigation of the total phenolic content and in vitro antioxidant activity of the various phenolic fractions was completed, to evaluate the potential of the extracts of these species for medicinal development. The free-radical-scavenging activities of the extracts derived from plants originating from Nanjing were proven to be higher than those of the other extracts, which correlated well with their total phenolic content.

Asymmetric synthesis of chloroisothreonine derivatives via syn-stereoselective Mannich-type additions across N-sulfinyl-α-chloroimines.

Mannich-type reactions of O-Boc glycolic esters across chiral N-sulfinyl-α-chloroaldimines resulted in the efficient and syn-stereoselective synthesis of new γ-chloro-α-hydroxy-ß-amino esters (dr > 99 : 1). The α-coordinating ability of the chlorine atom was of great importance for the diastereoselectivity of the Mannich-type reaction and overruled the chelation of the sulfinyl oxygen with the lithium ion of the incoming E-enolate in the transition state model. These novel chloroisothreonine derivatives proved to be excellent building blocks in asymmetric synthesis of novel syn-ß,γ-aziridino-α-hydroxy esters and biologically relevant trans-oxazolidinone carboxylic esters.

Haloperoxidase mediated quorum quenching by Nitzschia cf pellucida: study of the metabolization of N-acyl homoserine lactones by a benthic diatom.

Diatoms are known to produce a variety of halogenated compounds, which were recently shown to have a role in allelopathic interactions between competing species. The production of these compounds is linked to haloperoxidase activity. This research, has shown that this system may also be involved in diatom-bacteria interactions via the H2O2 dependent inactivation of a type of quorum sensing (QS) molecule, i.e., N-ß-ketoacylated homoserine lactones (AHLs), by a natural haloperoxidase system from the benthic diatom Nitzschia cf pellucida. The AHL degradation pathway towards corresponding halogenated derivatives was elucidated via HPLC-MS analysis and the synthesis of a broad series of novel halogenated AHL analogues as reference compounds. Furthermore, their biological activity as quorum sensing modulators was directly compared and evaluated against a series of naturally occurring ß-keto-AHLs. It has been demonstrated that the loss of the QS activity results from the final cleavage of the halogenated N-acyl chain of the signal molecules.

Synthesis and biological evaluation of novel N-α-haloacylated homoserine lactones as quorum sensing modulators.

Novel N-α-haloacylated homoserine lactones, in which a halogen atom was introduced at the α-position of the carbonyl function of the N-acyl chain, have been studied as quorum sensing (QS) modulators and compared with a library of natural N-acylated homoserine lactones (AHLs). The series of novel analogues consists of α-chloro, α-bromo and α-iodo AHL analogues. Furthermore, the biological QS activity of the synthetic AHL analogues compared to the natural AHLs was evaluated. Halogenated analogues demonstrated a reduced activity in the Escherichia coli JB523 bioassay, with the α-iodo lactones being the less active ones and the α-chloro AHLs the most potent QS agonists. Most of the α-haloacylated analogues did not exhibit a significant reduction when tested in the QS inhibition test. Therefore, these novel analogues could be utilized as chemical probes for QS structure-activity studies.

Synthesis and unambiguous NMR characterization of linear and branched N-alkyl chitosan derivatives.

Chitosan, sourced from abundant chitin-rich waste streams, emerges as a promising candidate in the realm of future functional materials and chemicals. While showing numerous advantageous properties, chitosan sometimes falls short of competing with today's non-renewable alternatives. Chemical derivatization, particularly through N-alkylation, proves promising in enhancing hydrophobic functionalities. This study synthesizes fifteen chitosan derivatives (degree of substitution = 2-10 %) using an improved reductive amination method. Next, selective depolymerization through acid hydrolysis reduced the chain rigidity imposed by the polymer backbone. This facilitated unambiguous structural characterization of the synthesized compounds using a combination of common NMR techniques. Two potential side reactions are identified for the first time, emphasizing the need for detailed structural information to unlock the true potential of these derivatives in future applications. HYPOTHESIS: The increase in chain mobility induced by the selective depolymerization of aliphatic N-alkyl chitosan derivatives allows for an unambiguous NMR characterization.

Diproline-induced resistance to parasitic nematodes in the same and subsequent rice generations: Roles of iron, nitric oxide and ethylene.

Induced resistance (IR) is a plant phenotype characterized by lower susceptibility to biotic challenges upon elicitation by so-called IR stimuli. Earlier, we identified diproline (cyclo(l-Pro-l-Pro)) as IR stimulus that protects rice (Oryza sativa) against the root-knot nematode Meloidogyne graminicola (Mg). In the current study, detailed transcriptome analyses at different time points, and under uninfected and nematode-infected conditions revealed that this rice IR phenotype is correlated with induction of genes related to iron (Fe), ethylene (ET) and reactive oxygen species (ROS)/reactive nitrogen species (RNS) metabolism. An infection experiment under Fe limiting conditions confirmed that diproline-IR is only effective under optimal Fe supply. Although total root Fe levels were not affected in diproline-treated plants, phytosiderophore secretion was found to be induced by this treatment. Experiments on mutant and transgenic rice lines impaired in ET or ROS/RNS metabolism confirmed that these metabolites are involved in diproline-IR. Finally, we provide evidence for transgenerational inheritance of diproline-IR (diproline-TIR), as two successive generations of diproline-treated ancestors exhibited an IR phenotype while themselves never being exposed to diproline. Transcriptome analyses on the offspring plants revealed extensive overlap between the pathways underpinning diproline-IR and diproline-TIR. Although diproline induces significant systemic changes in global DNA methylation levels early after treatment, such changes in DNA methylation were not detected in the descendants of these plants. To our knowledge, this is the first report of TIR in rice and the first transcriptional assessment of TIR in monocots.

Rearrangement of N-tert-butanesulfinyl α-halo imines with alkoxides to N-tert-butanesulfinyl 2-amino acetals as precursors of N-protected and N-unprotected α-amino carbonyl compounds.

Reaction of N-tert-butanesulfinyl α-halo imines with alkoxides afforded new N-tert-butanesulfinyl 2-amino acetals in good to excellent yield. These N-tert-butanesulfinyl 2-amino acetals are convenient precursors for the TMSOTf-promoted synthesis of the corresponding N-protected α-amino aldehydes and ketones, as well as for the HCl-promoted synthesis of 2-amino acetal hydrochlorides and α-amino ketone and α-amino aldehyde hydrochlorides in high yield. Via this method, an asymmetric synthesis of (S)-cathinone hydrochloride (er 94:6) was achieved.

Diastereoselective aldol reaction of zincated 3-chloro-3-methyl-1-azaallylic anions as key step in the synthesis of 1,2,3,4-tetrasubstituted 3-chloroazetidines.

Zincated 3-chloro-3-methyl-1-azaallylic anions undergo a stereoselective aldol addition across aromatic aldehydes and subsequent mesylation to produce syn α-chloro-ß-mesyloxyketimines, which were isolated in 80-84% yield and high diastereomeric excess (dr > 97/3) after purification via flash chromatography. The syn α-chloro-ß-mesyloxyketimines were further stereoselectively reduced to give stereochemically defined 3-aminopropyl mesylates, which were cyclized to 1,2,3,4-tetrasubstituted 3-chloroazetidines containing three contiguous stereogenic centers. DFT calculations on the key aldol addition revealed the presence of a highly ordered bimetallic six-membered twist-boat-like transition state structure with a tetra-coordinated metal cyclic structure. DFT calculations revealed that chelation of both zinc and lithium cations in the transition state structure leads to the experimentally observed high syn diastereoselectivity of aldol reactions.

Asymmetric synthesis of α,ß-diamino acid derivatives with an aziridine-, azetidine- and γ-lactone-skeleton via Mannich-type additions across α-chloro-N-sulfinylimines.

The efficient asymmetric synthesis of new chiral γ-chloro-α,ß-diamino acid derivatives via highly diastereoselective Mannich-type reactions of N-(diphenylmethylene) glycine esters across a chiral α-chloro-N-p-toluenesulfinylimine was developed. The influence of the base, LDA or LiHMDS, used for the formation of the glycine enolates, was of great importance for the anti-/syn-diastereoselectivity of the Mannich-type reaction. The γ-chloro-α,ß-diamino acid derivatives proved to be excellent building blocks for ring closure towards optically pure anti- and syn-ß,γ-aziridino-α-amino esters, and subsequent ring transformation into trans-3-aminoazetidine-2-carboxylic acid derivatives and α,ß-diamino-γ-butyrolactones.

Asymmetric synthesis of γ-chloro-α,ß-diamino- and ß,γ-aziridino-α-aminoacylpyrrolidines and -piperidines via stereoselective Mannich-type additions of N-(diphenylmethylene)glycinamides across α-chloro-N-sulfinylimines.

The asymmetric synthesis of new chiral γ-chloro-α,ß-diaminocarboxylamide derivatives by highly diastereoselective Mannich-type reactions of N-(diphenylmethylene)glycinamides across chiral α-chloro-N-p-toluenesulfinylaldimines was developed. The resulting (S(S),2S,3S)-γ-chloro-α,ß-diaminocarboxylamides were formed with the opposite enantiotopic face selectivity as compared to the (S(S),2R,3R)-γ-chloro-α,ß-diaminocarboxyl esters obtained via Mannich-type addition of analogous N-(diphenylmethylene)glycine esters across a chiral α-chloro-N-p-toluenesulfinylaldimine. Selective deprotection under different acidic reaction conditions and ring closure of the γ-chloro-α,ß-diaminocarboxylamides was optimized, which resulted in N(α)-deprotected syn-γ-chloro-α,ß-diaminocarboxylamides, N-sulfinyl-ß,γ-aziridino-α-aminocarboxylamide derivatives, a trans-imidazolidine, and an N(α),N(ß)-deprotected syn-γ-chloro-α,ß-diaminocarboxylamide.

Selection constrains lottery assembly in the microbiomes of closely related diatom species.

It is generally recognised that interactions between microalgae and bacteria play an important role in the functioning of marine ecosystems. In this context, increasing attention is paid to the processes that shape microalga-associated microbiomes. In recent years, conflicting evidence has been reported with respect to the relative importance of selective vs neutral processes in the assembly process. Whereas some studies report strong selection imposed by the host, others propose a more neutral, lottery-like assembly model according to which the chance of bacteria becoming part of the microbiome is proportional to their abundance in the environment and not driven by the selectional pressure created by the host. In the present study, we investigated to what degree selective vs neutral assembly processes constrain taxonomic, phylogenetic and functional variation within and between microbiomes associated with 69 isolates belonging to the Cylindrotheca closterium benthic marine diatom complex. The diatom cultures were initiated from non-axenic clonal isolates from different marine environments and geographic locations, and were then reared in a common garden (lab) environment. An important environmental imprint, likely due to in situ lottery dynamics, was apparent in the diatom microbiomes. However, microbiome assembly was also phylogenetically and functionally constrained through selective filtering related to the host microhabitat. Randomised microbiome assembly simulations revealed evidence for phylogenetic overdispersion in the observed microbiomes, reflecting an important role in the assembly process for competition between bacteria on the one hand and predominantly genetically driven differences between the hosts on the other hand. Our study thus shows that even between closely related diatom strains, host selection affects microbiome assembly, superimposing the predominantly stochastically driven recruitment process.

Belgian endive-derived biostimulants promote shoot and root growth in vitro.

Recovering biostimulant compounds from by-products of crops is a promising strategy to add value, enhance sustainability, and increase the environmental safety of the agricultural production chain. Here, we report consistent root and shoot growth-stimulating bioactivity present in water-based extracts from Belgian endive forced roots (Cichorium intybus var. foliosum) over two consecutive harvest years. The shoot and the primary root of in vitro cultivated Arabidopsis thaliana treated with Belgian endive extract were about 30% increased in size compared to plants grown under control conditions. The ornamental species Plectranthus esculentus also showed enhanced in vitro shoot and root growth, suggesting bioactivity on a broad range of species. Fractionation of the Belgian endive extracts into aqueous and organic subfractions coupled with bioactivity measurements showed that the principal root and shoot growth-promoting ingredients are primarily water-soluble. NMR-based characterization of the bioactive aqueous fractions revealed the presence of predominantly sugars and organic acids. Malate and sugars were abundant and common to all water fractions, suggesting these molecules contributed to the growth stimulation phenotype. The findings indicate that Belgian endive roots are a source for the development of organic waste-derived biostimulants with potential for application in tissue culture and putatively for soil-grown crop production.

Sunflower Bark Extract as a Biostimulant Suppresses Reactive Oxygen Species in Salt-Stressed Arabidopsis.

A survey of plant-based wastes identified sunflower (Helianthus annuus) bark extract (SBE), produced via twin-screw extrusion, as a potential biostimulant. The addition of SBE to Arabidopsis (Arabidopsis thaliana) seedlings cultured in vitro showed a dose-dependent response, with high concentrations causing severe growth inhibition. However, when priming seeds with SBE, a small but significant increase in leaf area was observed at a dose of 0.5 g of lyophilized powder per liter. This optimal concentration of SBE in the culturing medium alleviated the growth inhibition caused by 100 mM NaCl. The recovery in shoot growth was accompanied by a pronounced increase in photosynthetic pigment levels and a stabilization of osmotic homeostasis. SBE-primed leaf discs also showed a similar protective effect. SBE mitigated salt stress by reducing the production of reactive oxygen species (ROS) (e.g., hydrogen peroxide) by about 30% and developing more expanded true leaves. This reduction in ROS levels was due to the presence of antioxidative agents in SBE and by activating ROS-eliminating enzymes. Polyphenols, carbohydrates, proteins, and other bioactive compounds detected in SBE may have contributed to the cellular redox homeostasis in salt-stressed plants, thus promoting early leaf development by relieving shoot apical meristem arrest. Sunflower stalks from which SBE is prepared can therefore potentially be valorized as a source to produce biostimulants for improving salt stress tolerance in crops.