Development of an efficient and scalable bioprocess for the plant hormone 12-OPDA: Overcoming the hurdles of nature's biosynthesis.

Besides its native biological function as a plant hormone, cis-(+)-12-oxo-phytodienoic acid (12-OPDA) serves as a metabolite for the cellular formation of (-)-jasmonic acid and has also been shown to have an influence on mammalian cells. In order to make this biologically active, but at the same time very expensive natural product 12-OPDA broadly accessible for further biological and medicinal research, we developed an efficient bioprocess based on the utilization of a tailor-made whole-cell catalyst by following the principles of its biosynthesis in nature. After process optimization, the three-step one-pot synthesis of 12-OPDA starting from readily accessible α-linolenic acid could be conducted at appropriate technically relevant substrate loadings in the range of 5-20 g L-1. The desired 12-OPDA was obtained with an excellent conversion efficiency, and by means of the developed, efficient downstream-processing, this emulsifying as well as stereochemically labile biosynthetic metabolite 12-OPDA was then obtained with very high chemical purity (>99%) and enantio- and diastereomeric excess (>99% ee, 96% de) as well as negligible side-product formation (<1%). With respect to future technical applications, we also demonstrated the scalability of the production of the whole cell-biocatalyst in a high cell-density fermentation process.

Chemistry of Strigolactones, Key Players in Plant Communication.

Today, the use of artificial pesticides is questionable and the adaptation to global warming is a necessity. The promotion of favorable natural interactions in the rhizosphere offers interesting perspectives for changing the type of agriculture. Strigolactones (SLs), the latest class of phytohormones to be discovered, are also chemical mediators in the rhizosphere. We present in this review the diversity of natural SLs, their analogs, mimics, and probes essential for the biological studies of this class of compounds. Their biosynthesis and access by organic synthesis are highlighted especially concerning noncanonical SLs, the more recently discovered natural SLs. Organic synthesis of analogs, stable isotope-labeled standards, mimics, and probes are also reviewed here. In the last part, the knowledge about the SL perception is described as well as the different inhibitors of SL receptors that have been developed.

Chemical Synthesis of Triazole-Derived Suppressors of Strigolactone Functions.

Triazole is a five-membered heteroring consists of two carbon atoms and three nitrogen atoms and exhibits a wide range of biological activities. The basic heterocyclic rings are 1,2,3-triazole and 1,2,4-triazole. Here we describe the chemical synthetic methods for triazole derivatives that can suppress the function of SL by inhibiting SL biosynthesis pathway or SL perception sites such as D14.

Synthesis of Simple Strigolactone Mimics.

Strigolactones (SLs) are natural compounds occurring in plants which have a numerous functions in plant development; therefore, they are plant hormones. Unfortunately, their natural abundance is very low and because of their structure complexity it is difficult to prepare them in big quantities; alternatives with simpler structures and similar biological activity was developed. SLs mimics are compounds with simple synthesis. Methods for preparation of basic SLs mimics are described here.

Synthesis of Analogs of Strigolactones and Evaluation of Their Stability in Solution.

Strigolactones (SLs) are new plant hormones that play an important role in the control development of plants. They are germination stimulants for seed of parasitic weeds, are the branching factor of arbuscular mycorrhizal fungi and inhibitors for bud outgrowth and shoot branching. Natural SLs contain an annulated system of three rings (ABC scaffold) connected to a furanone (the D-ring) by an enol ether unit. The natural distribution of strigolactones is low, and their synthesis is long and difficult. Therefore, SL analogs are designed to have the same bioactiphore as natural SLs and an appreciable bioactivity. For the design a model is used based on the natural bioactiphore. Typical SL analogs are GR24, Nijmegen-1, and EM1 (derived from ethyl 2-phenylacetate). The synthesis of these SL analogs is reported together with their stability in aqueous solution.

Evaluation of the Effect of Strigolactones and Synthetic Analogs on Fungi.

Strigolactones (SLs) are components of root exudates as a consequence of active release from the roots into the soil. Notably, they have been described as stimulants of seed germination in parasitic plants and of the presymbiotic growth in arbuscular mycorrhizal (AM) fungi, which are a crucial component of the plant root beneficial microbiota. SLs have therefore the potential to influence other microbes that proliferate in the soil around the roots and may interact with plants. A direct effect of SL analogs on the in vitro growth of a number of saprotrophic or plant pathogenic fungi was indeed reported.Here we describe a standardized method to evaluate the effect of SLs or their synthetic analogs on AM and filamentous fungi. For AM fungi, we propose a spore germination assay since it is more straightforward than the hyphal branching assay and it does not require deep expertise and skills. For filamentous fungi that can grow in axenic cultures, we describe the assay based on SLs embedded in the solid medium or dissolved in liquid cultures where the fungus is inoculated to evaluate the effect on growth, hyphal branching or conidia germination. These assays are of help to test the activity of natural SLs as well as of newly designed SL analogs for basic and applied research.

Encapsulation of Synthesized Plant Growth Regulator Based on Copper(II) Complex in Chitosan/Alginate Microcapsules.

A new copper complex, trans-diaqua-trans-bis [1-hydroxy-1,2-di (methoxycarbonyl) ethenato] copper (abbreviation Cu(II) complex), was synthesized and its plant growth regulation properties were investigated. The results show a sharp dependence of growth regulation activity of the Cu(II) complex on the type of culture and its concentration. New plant growth regulator accelerated the development of the corn root system (the increase in both length and weight) but showed a smaller effect on the development of the wheat and barley root systems. Stimulation of corn growth decreased with increasing Cu(II) complex concentration from 0.0001% to 0.01% (inhibition at high concentrations-0.01%). The development of corn stems was also accelerated but to a lesser extent. Chitosan-coated calcium alginate microcapsules suitable for delivery of Cu(II) complex to plants were prepared and characterized. Analysis of the FTIR spectrum showed that complex molecular interactions between functional groups of microcapsule constituents include mainly electrostatic interactions and hydrogen bonds. Microcapsules surface exhibits a soft granular surface structure with substructures consisting of abundant smaller particles with reduced surface roughness. Release profile analysis showed Fickian diffusion is the rate-controlling mechanism of Cu(II) complex releasing. The obtained results give new insights into the complexity of the interaction between the Cu(II) complex and microcapsule formulation constituents, which can be of great help in accelerating product development for the application in agriculture.

Synthesis of New Brassinosteroid 24-Norcholane Type Analogs Conjugated in C-3 with Benzoate Groups.

The metabolism of brassinosteroid leads to structural modifications in the ring skeleton or the side alkyl chain. The esterification and glycosylation at C-3 are the most common metabolic pathways, and it has been suggested that conjugate brassinosteroids are less active or inactive. In this way, plants regulate the content of active brassinosteroids. In this work, the synthesis of brassinosteroid 24-norcholane type analogs conjugated at C-3 with benzoate groups, carrying electron donor and electron attractant substituents on the aromatic ring, is described. Additionally, their growth-promoting activities were evaluated using the Rice Lamina Inclination Test (RLIT) and compared with that exhibited by brassinolide (used as positive control) and non-conjugated analogs. The results indicate that at the lowest tested concentrations (10-8-10-7 M), all analogs conjugated at C-3 exhibit similar or higher activities than brassinolide, and the diasteroisomers with S configuration at C-22 are the more active ones. Increasing concentration (10-6 M) reduces the biological activities of analogs as compared to brassinolide.

Synthesis, Characterization, and Encapsulation of Novel Plant Growth Regulators (PGRs) in Biopolymer Matrices.

Novel plant growth regulators (PGRs) based on the derivatives of dehydroamino acids 2,3-dehydroaspartic acid dimethyl ester (PGR1), Z-isomer of the potassium salt of 2-amino-3-methoxycarbonylacrylic acid (PGR2) and 1-methyl-3-methylamino-maleimide (PGR3) have been synthesized and their growth-regulating properties investigated. Laboratory testing revealed their plant growth-regulating activity. PGR1 showing the most stimulating activity on all laboratory tested cultures were used in field experiments. Results showed that PGR1 is a highly effective environmentally friendly plant growth regulator with effects on different crops. Biopolymeric microcapsule formulations (chitosan/alginate microcapsule loaded with PGR) suitable for application in agriculture were prepared and characterized. Physicochemical properties and release profiles of PGRs from microcapsule formulations depend on the molecular interactions between microcapsule constituents including mainly electrostatic interactions and hydrogen bonds. The differences in the microcapsule formulations structure did not affect the mechanism of PGRs release which was identified as diffusion through microcapsules. The obtained results opened a perspective for the future use of microcapsule formulations as new promising agroformulations with a sustained and target release for plant growth regulation.

Synthesis of New Steroidal Carbamates with Plant-Growth-Promoting Activity: Theoretical and Experimental Evidence.

A priority of modern agriculture is to use novel and environmentally friendly plant-growth promoter compounds to increase crop yields and avoid the indiscriminate use of synthetic fertilizers. Brassinosteroids are directly involved in the growth and development of plants and are considered attractive candidates to solve this problem. Obtaining these metabolites from their natural sources is expensive and cumbersome since they occur in extremely low concentrations in plants. For this reason, much effort has been dedicated in the last decades to synthesize brassinosteroids analogs. In this manuscript, we present the synthesis and characterization of seven steroidal carbamates starting from stigmasterol, ß-sitosterol, diosgenin and several oxygenated derivatives of it. The synthesis route for functionalization of diosgenin included epoxidation and epoxy opening reactions, reduction of carbonyl groups, selective oxidation of hydroxyl groups, among others. All the obtained compounds were characterized by 1H and 13C NMR, HRMS, and their melting points are also reported. Rice lamina inclination test performed at different concentrations established that all reported steroidal carbamates show plant-growth-promoting activity. A molecular docking study evaluated the affinity of the synthesized compounds towards the BRI1-BAK1 receptor from Arabidopsis thaliana and three of the docked compounds displayed a binding energy lower than brassinolide.

3'-(Phenyl alkynyl) analogs of abscisic acid: synthesis and biological activity of potent ABA antagonists.

We report here the synthesis and biological testing of 3'-(phenyl alkynyl) abscisic ABA analogs, a new class of potent ABA antagonists. These ABA analogs incorporate a rigid framework of eight carbon atoms attached at the 3'-carbon atom of ABA that prevents folding of the ABA analog-bound receptor required for ABA signalling. The two-step synthesis is based upon the optimized conversion of natural (S)-ABA to 3'-iodo ABA which can be coupled to phenyl acetylenes using Sonogashira conditions, or to styryl compounds through Suzuki chemistry. The parent 3'-(phenyl alkynyl) ABA analog 7 was obtained in 29% yield, 74% yield based on recovered starting material. In a lentil seed germination assay, compound 7 was found to have more potent activity than other known 3'-substituted ABA antagonists to date. In a structure activity study parasubstituted phenyl alkynyl analogs had comparable activity to the analog 7 while the 3'-styryl ABA 18 was only slightly less active. Analog 7 overcame ABA inhibition of germination and seedling growth in a wide range of mono and dicot plant species, including canola, lentil, soybean, rice, wheat, barley, cannabis and canary seed. 3'-(Phenyl alkynyl) ABA analogs have numerous potential practical agricultural applications including promoting ripening of crops, dormancy breaking of seeds and woody perennials, as well as promoting seed germination, and growth under stress conditions as demonstrated in this report.

pH- and Time-Dependent Release of Phytohormones from Diruthenium Complexes.

The controlled release of functionally active compounds is important in a variety of applications. Here, we have synthesized, characterized, and studied the magnetic properties of three novel metal-metal-bonded tris(formamidinato) Ru25+ complexes. We have used different auxin-related hormones, indole-3-acetate (IAA), 2,4-dichlorophenoxyacetate (2,4-D), and 1-naphthaleneacetate (NAA), to generate [Ru2Cl(µ-DPhF)3(µ-IAA)] (RuIAA), [Ru2Cl(µ-DPhF)3(µ-2,4-D)] (Ru2,4-D), and [Ru2Cl(µ-DPhF)3(µ-NAA)] (RuNAA) (DPhF = N,N'-diphenylformamidinate). The crystal structures of RuIAA, RuIAA·THF, Ru2,4-D·CH2Cl2, and RuNAA·0.5THF have been determined by single-crystal X-ray diffraction. To assess the releasing capacity of the bound hormone, we have employed a biological assay that relied on Arabidopsis thaliana plants expressing an auxin reporter gene and we demonstrate that the release of the phytohormones from RuIAA, Ru2,4-D, and RuNAA is pH- and time-dependent. These studies serve as a proof of concept showing the potential of these types of compounds as biological molecule carriers.

p-Aminobenzoate Organic Salts as Potential Plant Growth Regulators for Tomatoes.

The discovery of environmentally friendly and inexpensive plant growth regulators (PGRs) for agronomically important crops is a necessity and must be considered a priority worldwide. This study provides the synthesis, structure determination and the biological evaluation of two binary organic salts as potential PGRs. New compounds have dual biological activity and are based on natural metabolite p-aminobenzoic acid (pABAH) and different alkanolamines. Studied compounds exhibit hydrogen-bonded 3D supramolecular architectures with different crystal packing due to the formation of one homosynthon and various heterosynthons. The biological profile of new compounds was investigated in laboratory and greenhouse on Solanum lycopersicum L., revealing the efficiency in promoting plant rooting and plant productivity. The results may have a positive impact on agricultural economics, developing new sustainable PGRs for tomatoes.

Curcumin-Cu(II) Ensemble-Based Fluorescence "Turn-On" Mode Sensing the Plant Defensive Hormone Salicylic Acid In Situ and In Vivo.

Salicylic acid (SA), a crucial, plant-derived signal molecule, is capable of launching global transcriptional reprogramming to assist plants in obtaining the systemic acquired resistance (SAR) mechanism. Thus, the accurate detection of SA will not only significantly contribute to the understanding of the plant SAR but also contribute to crop protection and to the security of the agricultural production and food supply. However, detection of SA using fluorescent probes is a great challenge for scientists, because SA analogues can significantly interfere with the detection results. Herein, we first reported using a simple, natural curcumin-Cu2+ ensemble to selectively and sensitively monitor SA in situ and in vivo, directed by a fluorescence "turn-on" mode. A binary combination curcumin-Cu2+ was first fabricated with a fluorescence "turn-off" pattern caused by the paramagnetic nature of Cu2+. Subsequently, a fluorescence "turn-on" response was performed for detecting SA accompanied by the formation of the ternary complex curcumin-Cu2+-SA due to the high affinity of SA toward Cu2+, which reduced the fluorescent impact caused by the paramagnetism of Cu2+. Further study revealed that the rationally designed hybrid probe could monitor SA in living cell lines. We anticipate that this finding can inspire the discovery of a high-performance SA probe.

Concise synthesis of heliolactone, a non-canonical strigolactone isolated from sunflower.

Heliolactone is one of the earliest identified non-canonical strigolactones. Its concise synthesis was achieved by employing Knoevenagel-type condensation and semi-reduction of a malonate intermediate as the key steps. This synthesis was performed in a non-stereoselective manner, and thus a racemic and diastereomeric mixture of heliolactone was obtained. The developed synthetic route is fairly concise and straightforward.

Synthesis and plant growth regulatory activities of 2',3'-PhABA and iso-2',3'-PhABA esters.

Methyl and phenyl esters of 2',3'-PhABA and iso-2',3'-PhABA were prepared for the biological investigation and development of practical applications. These esters exhibited excellent activity in most plant growth inhibitory assays. And, three esters were more efficient than ABA in stomatal closure. The 2',3'-PhABA analogs and their methyl esters have good stability in hydrolysis assay, and the different lipid solubility and permeability of different esters may be one of the origins of their active selectivity for different plants and physiological processes. Furthermore, in the study of drought tolerance, all four esters had comparable activity to ABA. These results suggest that these esters were potent plant growth regulator (PGR) candidates for anti-drought. The finding that different esters have different selective bioactivity and biophysical properties indicates that these esters not only function as ABA-like PGRs but also have the possibility as potential selective pro-hormone. 2',3'-BenzoABA esters as PGR candidates with prolonged and selective bioactivity.

Effect of prohydrojasmon on total phenolic content, anthocyanin accumulation and antioxidant activity in komatsuna and lettuce.

Prohydrojasmon has been reported to improve the quality of crops. However, most previous studies have investigated its application on fruits. Here, we evaluated the effect of prohydrojasmon on the growth and total phenolic content, anthocyanin content, and antioxidant activity in komatsuna (Brassica rapa var. periviridis) and lettuce (Lactuca sativa L.). Prohydrojasmon did not show any serious inhibitory effect. Prohydrojasmon applied to komatsuna at a concentration of 0.5 µM significantly increased the total phenolic content and anthocyanin content, and a concentration of 1 µM increased the antioxidant activity. In lettuce, prohydrojasmon at a concentration of 400 µM significantly increased the total phenolic content and anthocyanin content, while a concentration of 0.5 µM significantly increased the antioxidant activity. These results suggest that prohydrojasmon positively affects the phenolic compound and anthocyanin accumulation and antioxidant activity in komatsuna and lettuce without adversely affecting growth.

New aromatic 6-substituted 2'-deoxy-9-(ß)-d-ribofuranosylpurine derivatives as potential plant growth regulators.

Cytokinins are naturally occurring substances that act as plant growth regulators promoting plant growth and development, including shoot initiation and branching, and also affecting apical dominance and leaf senescence. Aromatic cytokinin 6-benzylaminopurine (BAP) has been widely used in micropropagation systems and biotechnology. However, its 9-glucoside (BAP9G) accumulates in explants, causing root inhibition and growth heterogenity. To overcome BAP disadvantages, a series of ring-substituted 2'-deoxy-9-(ß)-d-ribofuranosylpurine derivatives was prepared and examined in different classical cytokinin bioassays. Amaranthus, senescence and tobacco callus bioassays were employed to provide details of cytokinin activity of 2'-deoxy-9-(ß)-d-ribosides compared to their respective free bases and ribosides. The prepared derivatives were also tested for their recognition by cytokinin receptors of Arabidopsis thaliana AHK3 and CRE1/AHK4. The ability of aromatic N6-substituted adenine-2'-deoxy-9-(ß)-d-ribosides to promote plant growth and delay senescence was increased considerably and, in contrast to BAP, no loss of cytokinin activity at higher concentrations was observed. The presence of a 2'-deoxyribosyl moiety at the N9-position led to an increase in cytokinin activities in comparison to the free bases and ribosides. The antioxidant capacity, cytotoxicity and effect on the MHV-68 gammaherpesvirus strain were also examined.

Quaternary Ammonium Salts of Iminofullerenes: Fabrication and Effect on Seed Germination.

In this study, novel water-soluble quaternary ammonium salts of iminofullerenes (IFQA) were synthesized by nitrene chemistry in combination with quaternization and identified as [C60(NCH2CH2NH3+·CF3COO-)4·10H2O]n by various spectroscopies. Maize and Arabidopsis seeds were used to test the bioactivity of IFQA in seed germination. Compared with the control, maize seed exposure to 50 mg/L IFQA (normal: 73.1% vs 58.7%; drought: 66.7% vs 50.0% at the second day) and Arabidopsis seed exposure to 20 mg/L IFQA (normal: 77.5% vs 58.8%; drought: 63.3% vs 36.7% at the second day) had higher germination rates and quicker germination. The results of two-dimensional gel electrophoresis combined with mass spectroscopy showed that the abundance of 21 proteins in embryo proteome of maize seeds was significantly changed (>1.5 fold). The downregulated six storage proteins and upregulated four proteins induced by IFQA for energy production and sugar metabolism indicated a faster metabolic activity of maize seed germination. The upregulated eight stress-related proteins and antioxidant enzymes suggested that the role of IFQA was to activate the metabolic processes in seed germination and also increase seed stress response. The results provide important information to understand the mechanism of seed germination enhancement by carbon nanomaterials.

Strigolactones: Plant Hormones with Promising Features.

Almost 80 years after the discovery of the first plant hormone, auxin, a few years ago a new class of plant hormones, the strigolactones, was discovered. These molecules have unprecedented biological activity in a number of highly important biological processes in plants but also outside the plant in the rhizosphere, the layer of soil surrounding the roots of plants and teeming with life. The exploitation of this amazing biological activity is not without challenges: the synthesis of strigolactones is complicated and designing the desired activity a difficult task. This minireview describes the current state of knowledge about the strigolactones and how synthetic analogs can be developed that can potentially contribute to the development of a sustainable agriculture.