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Practical guide toward discovery of biomolecules with biostimulant activity

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Abstract

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.

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... This unsustainable approach has underlined the need for more sustainable agricultural practices promoting biodiversity, which will be critical for food production and quality in the future, especially in the climate change context [5,6]. As one of the more promising farming models, the RA model practice has been introduced alongside other sustainable initiatives [7]. ...
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Plants require solar energy to grow through oxygenic photosynthesis. However, when light intensity exceeds the optimal range for photosynthesis it causes abiotic stress and physiological damage in plants. In response to high light stress, plants initiate a series of signal transduction from chloroplasts to whole cells and from locally stressed tissues to the rest of the plant body. These signals trigger a variety of physiological and biochemical reactions intended to mitigate the deleterious effects of high light intensity such as photodamage and photoinhibition. Light stress protection mechanisms include chloroplastic Reactive Oxygen Species (ROS) scavenging, chloroplast and stomatal movement, and anthocyanin production. Photosynthetic apparatuses, being the direct targets of photodamage, have also developed various acclimation processes such as thermal energy dissipation through Non-Photochemical Quenching (NPQ), photorepair of Photosystem II (PSII), and transcriptional regulation of photosynthetic proteins. Fluctuating light is another mild but persistent type of light stress in nature, which has unfortunately been poorly investigated. Current studies suggest however that state transitions and cyclic electron transport are the main adaptive mechanisms for mediating fluctuating light stress in plants. Here, we review the current breadth of knowledge regarding physiological and biochemical responses to both high light stress and fluctuating light stress.
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Owing to their unique abilities to manipulate, label, and image individual molecules in vitro and in cellulo, single-molecule techniques provide previously unattainable access to elementary biological processes. In imaging, single-molecule fluorescence resonance energy transfer (smFRET) and protein-induced fluorescence enhancement in vitro can report on conformational changes and molecular interactions, single-molecule pull-down (SiMPull) can capture and analyze the composition and function of native protein complexes, and single-molecule tracking (SMT) in live cells reveals cellular structures and dynamics. In labeling, the abilities to specifically label genomic loci, mRNA, and nascent polypeptides in cells have uncovered chromosome organization and dynamics, transcription and translation dynamics, and gene expression regulation. In manipulation, optical tweezers, integration of single-molecule fluorescence with force measurements, and single-molecule force probes in live cells have transformed our mechanistic understanding of diverse biological processes, ranging from protein folding, nucleic acids-protein interactions to cell surface receptor function.
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Fluorescent 1 : 1, 1 : 2 and 1 : 3 sugar-O-BODIPY conjugates of glucose, xylose and ribose were characterised by ¹ H– ¹¹ B HMBC and ¹¹ B NMR to discriminate between boron bound to 1,2-, 1,3- or 1,4-diol sites and furanose/pyranose sugar forms.
Chapter
Biological macromolecules are large cellular components abundantly obtained naturally and are responsible for varieties of essential functions for the growth and survival of living organisms. There are four important classes of biological macromolecules, viz., carbohydrates, lipids, proteins, and nucleic acids. These possess some favorable characteristics, such as good biocompatibility, excellent biodegradability, desired mechanical strength, better bioavailability, etc., and these characteristics are directing the uses of these biological macromolecules in the biomedical and related fields. Biological macromolecules can also modulate the pathophysiology of neurodegenerative disorders/diseases. The current chapter deals with a brief discussion about the sources, properties, and valued applications of various biological macromolecules.
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Plants use electrical and chemical signals for systemic communication. Herbivory, for instance, appears to trigger local apoplasmic glutamate accumulation, systemic electrical signals and calcium waves that travel to report insect damage to neighboring leaves and initiate defense. To monitor extra‐ and intracellular glutamate concentrations in plants, we generated Arabidopsis lines expressing genetically encoded fluorescent glutamate sensors. In contrast to cytosolically localized sensors, extracellularly displayed variants inhibited plant growth and proper development. Phenotypic analyses of high‐affinity display sensor lines revealed that root meristem development, particularly the quiescent center (QC), number of lateral roots, vegetative growth and floral architecture were impacted. Notably, the severity of the phenotypes was positively correlated with the affinity of the display sensors, intimating that their ability to sequester glutamate at the surface of the plasma membrane was responsible for the defects. Root growth defects were suppressed by supplementing culture media with low levels of glutamate. Together, the data indicate that sequestration of glutamate at the cell surface either disrupts the supply of glutamate to meristematic cells and/or impairs localized glutamatergic signaling important for developmental processes.
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Aerobic composting is a typical biochemical process of stabilization and harmlessness of organic wastes during which organic matter degrades, and then aggregates, to produce humic substances (HSs). HSs are a core product of—and a crucial indicator of—the maturation of compost that can be used in soil amendments. The formation of HSs is affected by the characteristics of the raw materials involved, the presence of compost additives, microbial activity, temperature, pH, the C/N ratio, moisture content, oxygen content and particle size, all of which can interact with each other. The formation of HSs is therefore complex. Moreover, it is difficult to identify definitive structures of humic acids (HAs) and fulvic acids (FAs), which are the two major components of HSs. However, HSs represent the same functional groups and structural arrangements, which helps to predict their structures. Functional groups represented by phenol and carboxylic acid groups of HAs and FAs can provide various agronomic functions, such as plant growth enhancement, water and nutrient retention, and disease suppression capacity. Overall, HSs can act as a soil amendment, fertilizer, and plant growth regulator. These functions of HSs enhance the reuse potential of organic waste compost products; however, this requires scientific control of various composting parameters and appropriate application of final products.
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Background Spices are rich source of naturally occurring bioactives and are used in different food applications. Major spice bioactives include essential oils, oleoresins, polysaccharides, phenolics, alkaloids, vitamins, and carotenoids. Conventional extraction of these bioactives has been associated with a high requirement of time, solvent, and energy which diminishes the application. Scope and approach This review discusses and summaries the recent findings on UAE for extraction of bioactives from spices. The concepts, mechanisms, factors affecting the sonication process, and their application in the extraction of spice bioactives are reviewed. The integration of UAE with other emerging technologies, mainly microwave-assisted and supercritical fluid (CO2) assisted extraction methods in the field of spice bioactives extraction are also explored. A comparison of different optimization and modeling methods used for UAE of spices are discussed and enunciate the gaps and possible future trend. Key findings and conclusion Acoustic cavitation generated during ultrasound treatment produces shock waves, microjets, shear force, and sonochemical reactions causing plant cell erosion and pore formation. It enhances mass transfer paving way for application of green solvents such as water. Further the Two-site kinetic model and Peleg's model are most versatile in explaining the UAE of bioactives from spices. Maximized recovery of bioactives, could be attained by selecting combination of technologies and process parameters for improved energy efficiency reduced time and energy consumption.
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The tools available to carry out in vivo analysis of Ca2+ dynamics in plants are powerful and mature technologies that still require the proper controls.
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Biological processes are highly dynamic, and during plant growth, development, and environmental interactions, they occur and influence each other on diverse spatiotemporal scales. Understanding plant physiology on an organismic scale requires analyzing biological processes from various perspectives, down to the cellular and molecular levels. Ideally, such analyses should be conducted on intact and living plant tissues. Fluorescent protein-based in vivo biosensing using genetically encoded fluorescent indicators (GEFIs) is a state-of-the-art methodology for directly monitoring cellular ion, redox, sugar, hormone, ATP and phosphatidic acid dynamics, and protein kinase activities in plants. The steadily growing number of diverse but technically compatible genetically encoded biosensors, the development of dual-reporting indicators, and recent achievements in plate-reader-based analyses now allow for GEFI multiplexing: the simultaneous recording of multiple GEFIs in a single experiment. This in turn enables in vivo multiparameter analyses: the simultaneous recording of various biological processes in living organisms. Here we provide an update on currently established direct fluorescent protein-based biosensors in plants, discuss their functional principles, and highlight important biological findings accomplished by employing various approaches of GEFI-based multiplexing. We also discuss challenges and provide advice for fluorescent protein-based biosensor analyses in plants.
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Fluorescence is widely used to detect functional groups and ions, and peptides are used in various fields due to their excellent biological activity. In recent years, research on fluorescent amino acids has intensified, especially with the natural fluorescent amino acids such as tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe). They use their respective fluorophores to undergo π-π transition under excitation with light of a particular wavelength, and then release a large number of photons, which produce fluorescence. Thus, they are widely used as building units to enhance the fluorescence properties of other molecules. Peptide-based fluorescence technology has expanded the research in the field of biochemistry. Peptide-based fluorescence involving fluorescent amino acids not only retains the biocompatibility of peptides, but also relies on their fluorescent groups to enhance the fluorescent properties of the peptide itself, breaking the original limitations and connecting different fields to complement each other. This review summarizes the luminescence mechanism and fluorescent properties of the natural fluorescent amino acids and focuses on the fluorescence emission of peptides embedded with these fluorescent amino acids or fluorophores. Finally, we summarize the applications of peptide-based fluorescence in sensing drug release, metal ions and biomolecules, and present new challenges and expectations for the future development in this field. © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021.
Article
The pH parameter of soil plays a key role for plant nutrition as it is affecting the availability of minerals and consequently determines plant growth. Although the mechanisms by which root perceive the external pH is still unknown, the impact of external pH on tissue growth has been widely studied especially in hypocotyl and root. Thanks to technological development of cell imaging and fluorescent sensors, we can now monitor pH in real time with at subcellular definition. In this focus, fluorescent dye-based, as well as genetically-encoded pH indicators are discussed especially with respect to their ability to monitor acidic pH in the context of primary root. The notion of apoplastic subdomains is discussed and suggestions are made to develop fluorescent indicators for pH values below 5.0.