Most cited article - PubMed ID 30271419
Characterization of Biostimulant Mode of Action Using Novel Multi-Trait High-Throughput Screening of Arabidopsis Germination and Rosette Growth
N-Sulfonated IAA was discovered as a novel auxin metabolite in Urtica where it is biosynthesized de novo utilizing inorganic sulfate. It showed no auxin activity in DR5::GUS assay, implying possible inactivation/storage mechanism. A novel auxin derivative, N-sulfoindole-3-acetic acid (IAA-N-SO3H, SIAA), was discovered in stinging nettle (Urtica dioica) among 116 sulfonated metabolites putatively identified by a semi-targeted UHPLC-QqTOF-MS analysis of 23 plant/algae/fungi species. These sulfometabolites were detected based on the presence of a neutral loss of sulfur trioxide, as indicated by the m/z difference of 79.9568 Da in the MS2 spectra. The structure of newly discovered SIAA was confirmed by synthesizing its standard and comparing retention time, m/z and MS2 spectrum with those of SIAA found in Urtica. To study its natural occurrence, 73 species in total were further analyzed by UHPLC-QqTOF-MS or targeted UHPLC-MS/MS method with a limit of detection of 244 fmol/g dry weight. However, SIAA was only detected in Urtica at a concentration of 13.906 ± 9.603 nmol/g dry weight. Its concentration was > 30 times higher than that of indole-3-acetic acid (IAA), and the SIAA/IAA ratio was further increased under different light conditions, especially in continuous blue light. In addition to SIAA, structurally similar metabolites, N-sulfoindole-3-lactic acid, 4-(sulfooxy)phenyllactic acid and 4-(sulfooxy)phenylacetic acid, were detected in Urtica for the first time. SIAA was biosynthesized from inorganic sulfate in seedlings, as confirmed by the incorporation of exogenous 34S-ammonium sulfate (1 mM and 10 mM). SIAA exhibited no auxin activity, as demonstrated by both the Arabidopsis DR5::GUS assay and the Arabidopsis phenotype analysis. Sulfonation of IAA may therefore be a mechanism for IAA deactivation and/or storage in Urtica, similar to sulfonation of the jasmonates in Arabidopsis.
- Keywords
- N-Sulfoindole-3-acetic acid, Indole-3-acetic acid, Mass spectrometry, Metabolomics, Phytohormone, Sulfonated,
- MeSH
- Arabidopsis metabolism MeSH
- Indoleacetic Acids * metabolism MeSH
- Plant Growth Regulators metabolism MeSH
- Tandem Mass Spectrometry MeSH
- Urtica dioica metabolism MeSH
- Chromatography, High Pressure Liquid MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- indoleacetic acid MeSH Browser
- Indoleacetic Acids * MeSH
- Plant Growth Regulators MeSH
Putrescine (Put) is a promising small molecule-based biostimulant to enhance plant growth and resilience, though its mode of action remains unclear. This study investigated the Put priming effect on Arabidopsis mutant lines (Atadc1, Atadc2, Atnata1, and Atnata2) under control conditions and salinity to understand its role in regulating plant growth. The Atadc2 mutant, characterized by reduced endogenous Put levels, showed insensitivity to Put priming without growth enhancement, which was linked to significant imbalances in nitrogen metabolism, including a high Gln/Glu ratio. Contrarily, the Atnata2 mutant exhibited significant growth improvement and upregulated AtADC2 expression, particularly under Put priming, highlighting these genes' involvement in regulating plant development. Put priming enhanced plant growth by inducing the accumulation of specific polyamines (free, acetylated, conjugated, or bound form) and improving light-harvesting efficiency, particularly in the Atnata2 line. Our findings suggest that AtNATA2 may negatively regulate Put synthesis and accumulation via AtADC2 in the chloroplast, impacting light harvesting in photosystem II (PSII). Furthermore, the Atadc2 mutant line exhibited upregulated AtADC1 but reduced AcPut levels, pointing to a cross-regulation among these genes. The regulation by AtNATA2 on AtADC2 and AtADC2 on AtADC1 could be crucial for plant growth and overall stress tolerance by interacting with polyamine catabolism, which shapes the plant metabolic profile under different growth conditions. Understanding the regulatory mechanisms involving crosstalk between AtADC and AtNATA genes in polyamine metabolism and the connection with certain SMBBs like Put can lead to more effective agricultural practices, improving plant growth, nitrogen uptake, and resilience under challenging conditions.
- MeSH
- Arabidopsis * genetics growth & development physiology metabolism MeSH
- Photosystem II Protein Complex metabolism MeSH
- Loss of Function Mutation MeSH
- Polyamines metabolism MeSH
- Arabidopsis Proteins * genetics metabolism MeSH
- Putrescine * metabolism MeSH
- Gene Expression Regulation, Plant * MeSH
- Salt Tolerance * genetics MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Photosystem II Protein Complex MeSH
- Polyamines MeSH
- Arabidopsis Proteins * MeSH
- Putrescine * MeSH
Commercial interest in biostimulants as a tool for sustainable green economics and agriculture concepts is on a steep rise, being followed by increasing demand to employ efficient scientific methods to develop new products and understand their mechanisms of action. Biostimulants represent a highly diverse group of agents derived from various natural sources. Regardless of their nutrition content and composition, they are classified by their ability to improve crop performance through enhanced nutrient use efficiency, abiotic stress tolerance, and quality of crops. Numerous reports have described modern, non-invasive sensor-based phenotyping methods in plant research. This review focuses on applying phenotyping approaches in biostimulant research and development, and maps the evolution of interaction of these two intensively growing domains. How phenotyping served to identify new biostimulants, the description of their biological activity, and the mechanism/mode of action are summarized. Special attention is dedicated to the indoor high-throughput methods using model plants suitable for biostimulant screening and developmental pipelines, and high-precision approaches used to determine biostimulant activity. The need for a complex method of testing biostimulants as multicomponent products through integrating other -omic approaches followed by advanced statistical/mathematical tools is emphasized.
- Keywords
- -omics, High-throughput screening, mechanism of action, mode of action, plant biostimulants, plant breeding, plant phenotyping, sensors,
- MeSH
- Stress, Physiological * MeSH
- Research MeSH
- Crops, Agricultural * MeSH
- Agriculture methods MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
Biostimulants have become an asset for agriculture since they are a greener alternative to traditionally used plant protection products. Also, they have gained the farmers' acceptance due to their effect on enhancing the plant's natural defense system against abiotic stresses. Besides commercially available complex products, small molecule-based biostimulants are useful for industry and research. Among them, polyamines (PAs) are well-studied natural compounds that can elicit numerous positive responses in drought-stressed plants. However, the studies are merely focused on the vegetative development of the plant. Therefore, we aimed to evaluate how drenching with putrescine (Put) and spermidine (Spd) modified the maize production and the yield quality parameters. First, a dosage optimization was performed, and then the best PA concentrations were applied by drenching the maize plants grown under well-watered (WW) conditions or water deficit (WD). Different mechanisms of action were observed for Put and Spd regarding maize production, including when both PAs similarly improved the water balance of the plants. The application of Put enhanced the quality and quantity of the yield under WW and Spd under WD. Regarding the nutritional quality of the grains, both PAs increased the carbohydrates content, whereas the contribution to the protein content changed by the interaction between compound and growth conditions. The mineral content of the grains was also greatly affected by the water condition and the PA application, with the most relevant results observed when Spd was applied, ending with flour richer in Zn, Cu, and Ca minerals that are considered important for human health. We showed that the exogenous PA application could be a highly efficient biofortification approach. Our findings open a new exciting use to be studied deep in the biostimulant research.
- Keywords
- Zea mays, drenching, mineral nutrition, polyamines, yield,
- Publication type
- Journal Article MeSH
The application of biostimulants appears to be an environmentally friendly, innovative, and sustainable agronomical tool to mitigate the negative effects induced by adverse climatology in traditional grape-growing regions such as La Rioja (Spain). However, their mechanism of action in grapevines is still unclear. We evaluated how commercial substances (two from Ascophyllum nodosum extraction and one amino acids-based biostimulant) and the non-proteinogenic amino acid β-aminobutyric acid (BABA) affect the quality and quantity of musts and grapes in Vitis vinifera L. cv. Tempranillo from a semi-arid region of La Rioja during two seasons. We hypothesized an enhancement in organic metabolites in berries and leaves in response to these treatments, changing the organoleptic characteristics of the final products. The treatments altered the primary metabolites such as carbohydrates, organic acids (AcOrg), and free amino acids, first in the leaves as the effect of the foliar application and second in grapes and musts. As the main result, the biostimulant efficiency depended on the climatology and vineyard location to improve the final yield. Whereas biostimulant application enhanced the yield in 2018 (less dry year), it did not help production in 2019 (dry year). BABA was the most efficient biostimulant, enhancing plant production. Regarding yield quality, the biostimulant application improved the musts mainly by enhancing the fumaric acid content and by reducing carbohydrates, except in BABA-treated plants, where they were accumulated. These results corroborate biostimulants as an exciting approach in wine production, especially for improving wine quality.
- Keywords
- Vitis vinifera L., biostimulants, grapevine, growth stages, phenology, primary metabolism, water deficit,
- Publication type
- Journal Article MeSH
Biostimulants became a hotspot in the fight to alleviate the consequences of abiotic stresses in crops. Due to their complex nature, it is challenging to obtain stable and reproducible final products and more challenging to define their mechanism of action. As an alternative, small molecule-based biostimulants, such as polyamines have promoted plant growth and improved stress tolerance. However, profound research about their mechanisms of action is still missing. To go further, we tested the effect of putrescine (Put) and its precursor ornithine (Orn) and degradation product 1,3-diaminopropane (DAP) at two different concentrations (0.1 and 1 mM) as a seed priming on in vitro Arabidopsis seedlings grown under optimal growth conditions, osmotic or salt stress. None of the primings affected the growth of the seedlings in optimal conditions but altered the metabolism of the plants. Under stress conditions, almost all primed plants grew better and improved their greenness. Only Orn-primed plants showed different plant responses. Interestingly, the metabolic analysis revealed the implication of the N- acetylornithine and Orn and polyamine conjugation as the leading player regulating growth and development under control and stress conditions. We corroborated polyamines as very powerful small molecule-based biostimulants to alleviate the adverse abiotic stress effects.
- Keywords
- abiotic stress, biostimulant, growth, plant phenotyping,
- Publication type
- Journal Article MeSH
The use of plant biostimulants contributes to more sustainable and environmentally friendly farming techniques and offers a sustainable alternative to mitigate the adverse effects of stress. Protein hydrolysate-based biostimulants have been described to promote plant growth and reduce the negative effect of abiotic stresses in different crops. However, limited information is available about their mechanism of action, how plants perceive their application, and which metabolic pathways are activating. Here we used a multi-trait high-throughput screening approach based on simple RGB imaging and combined with untargeted metabolomics to screen and unravel the mode of action/mechanism of protein hydrolysates in Arabidopsis plants grown in optimal and in salt-stress conditions (0, 75, or 150 mM NaCl). Eleven protein hydrolysates from different protein sources were used as priming agents in Arabidopsis seeds in three different concentrations (0.001, 0.01, or 0.1 μl ml-1). Growth and development-related traits as early seedling establishment, growth response under stress and photosynthetic performance of the plants were dynamically scored throughout and at the end of the growth period. To effectively classify the functional properties of the 11 products a Plant Biostimulant Characterization (PBC) index was used, which helped to characterize the activity of a protein hydrolysate based on its ability to promote plant growth and mitigate stress, and to categorize the products as plant growth promoters, growth inhibitors and/or stress alleviator. Out of 11 products, two were identified as highly effective growth regulators and stress alleviators because they showed a PBC index always above 0.51. Using the untargeted metabolomics approach, we showed that plants primed with these best performing biostimulants had reduced contents of stress-related molecules (such as flavonoids and terpenoids, and some degradation/conjugation compounds of phytohormones such as cytokinins, auxins, gibberellins, etc.), which alleviated the salt stress response-related growth inhibition.
Plant phenomics is becoming a common tool employed to characterize the mode of action of biostimulants. A combination of this technique with other omics such as metabolomics can offer a deeper understanding of a biostimulant effect in planta. However, the most challenging part then is the data analysis and the interpretation of the omics datasets. In this work, we present an example of how different tools, based on multivariate statistical analysis, can help to simplify the omics data and extract the relevant information. We demonstrate this by studying the effect of protein hydrolysate (PH)-based biostimulants derived from different natural sources in lettuce and tomato plants grown in controlled conditions and under salinity. The biostimulants induced different phenotypic and metabolomic responses in both crops. In general, they improved growth and photosynthesis performance under control and salt stress conditions, with better performance in lettuce. To identify the most significant traits for each treatment, a random forest classifier was used. Using this approach, we found out that, in lettuce, biomass-related parameters were the most relevant traits to evaluate the biostimulant mode of action, with a better response mainly connected to plant hormone regulation. However, in tomatoes, the relevant traits were related to chlorophyll fluorescence parameters in combination with certain antistress metabolites that benefit the electron transport chain, such as 4-hydroxycoumarin and vitamin K1 (phylloquinone). Altogether, we show that to go further in the understanding of the use of biostimulants as plant growth promotors and/or stress alleviators, it is highly beneficial to integrate more advanced statistical tools to deal with the huge datasets obtained from the -omics to extract the relevant information.
Drought and salinity reduce seed germination, seedling emergence, and early seedling establishment, affect plant metabolism, and hence, reduce crop yield. Development of technologies that can increase plant tolerance of these challenging growth conditions is a major current interest among plant scientists and breeders. Seed priming has become established as one of the practical approaches that can alleviate the negative impact of many environmental stresses and improve the germination and overall performance of crops. Hormopriming using different plant growth regulators has been widely demonstrated as effective, but information about using cytokinins (CKs) as priming agents is limited to only a few studies using kinetin or 6-benzylaminopurine (BAP). Moreover, the mode of action of these compounds in improving seed and plant fitness through priming has not yet been studied. For many years, BAP has been one of the CKs most commonly applied exogenously to plants to delay senescence and reduce the impact of stress. However, rapid endogenous N 9-glucosylation of BAP can result in negative effects. This can be suppressed by hydroxylation of the benzyl ring or by appropriate N 9 purine substitution. Replacement of the 2' or 3' hydroxyl groups of a nucleoside with a fluorine atom has shown promising results in drug research and biochemistry as a means of enhancing biological activity and increasing chemical or metabolic stability. Here, we show that the application of this chemical modification in four new N 9-substituted CK derivatives with a fluorinated carbohydrate moiety improved the antisenescence properties of CKs. Besides, detailed phenotypical analysis of the growth and development of Arabidopsis plants primed with the new CK analogs over a broad concentration range and under various environmental conditions revealed that they improve growth regulation and antistress activity. Seed priming with, for example, 6-(3-hydroxybenzylamino)-2'-deoxy-2'-fluoro-9-(β)-D-arabinofuranosylpurine promoted plant growth under control conditions and alleviated the negative effects of the salt and osmotic stress. The mode of action of this hormopriming and its effect on plant metabolism were further analyzed through quantification of the endogenous levels of phytohormones such as CKs, auxins and abscisic acid, and the results are discussed.
- Keywords
- Arabidopsis, abiotic stress, antisenescence, cytokinin analogs, hormopriming, plant biostimulant characterization index,
- Publication type
- Journal Article MeSH
Designing and developing new biostimulants is a crucial process which requires an accurate testing of the product effects on the morpho-physiological traits of plants and a deep understanding of the mechanism of action of selected products. Product screening approaches using omics technologies have been found to be more efficient and cost effective in finding new biostimulant substances. A screening protocol based on the use of high-throughput phenotyping platform for screening new vegetal-derived protein hydrolysates (PHs) for biostimulant activity followed by a metabolomic analysis to elucidate the mechanism of the most active PHs has been applied on tomato crop. Eight PHs (A-G, I) derived from enzymatic hydrolysis of seed proteins of Leguminosae and Brassicaceae species were foliarly sprayed twice during the trial. A non-ionic surfactant Triton X-100 at 0.1% was also added to the solutions before spraying. A control treatment foliarly sprayed with distilled water containing 0.1% Triton X-100 was also included. Untreated and PH-treated tomato plants were monitored regularly using high-throughput non-invasive imaging technologies. The phenotyping approach we used is based on automated integrative analysis of photosynthetic performance, growth analysis, and color index analysis. The digital biomass of the plants sprayed with PH was generally increased. In particular, the relative growth rate and the growth performance were significantly improved by PHs A and I, respectively, compared to the untreated control plants. Kinetic chlorophyll fluorescence imaging did not allow to differentiate the photosynthetic performance of treated and untreated plants. Finally, MS-based untargeted metabolomics analysis was performed in order to characterize the functional mechanisms of selected PHs. The treatment modulated the multi-layer regulation process that involved the ethylene precursor and polyamines and affected the ROS-mediated signaling pathways. Although further investigation is needed to strengthen our findings, metabolomic data suggest that treated plants experienced a metabolic reprogramming following the application of the tested biostimulants. Nonetheless, our experimental data highlight the potential for combined use of high-throughput phenotyping and metabolomics to facilitate the screening of new substances with biostimulant properties and to provide a morpho-physiological and metabolomic gateway to the mechanisms underlying PHs action on plants.
