TBS-pyrrole as an "universal" reference to quantify artemisinin and structurally-diverse natural products in plants extracts by NMR.

The commercial production of artemisinin and other valuable bioactive natural products depends on their plant sources, which may provide variable amounts of the compound depending on plant variety, the period of the year, abiotic stress and other factors. Therefore, it requires a method for large-scale, low-cost natural product quantification. The standard HPLC and UHPLC methods are accurate but the analysis are costly and require different optimization for structurally-diverse products. An alternative method using NMR with TBS-pyrrole as a novel "universal" reference affords a simple, fast method to quantify many different products. The method is shown with antimalarial artemisinin, whose yield using conventional and novel extraction procedures was determined by standard UHPLC-MS procedures and by our NMR protocol, with similar quantification results. The novel reference compound does not interfere with artemisinin or extract signals, only needs a small amount of the extract, is accurate and operationally simple, and a large volume of samples can be processed in little time. Moreover, bioactive terpenes, steroids, alkaloids, aromatic compounds, and quinones, among others, were quantified in a model vegetal extract with this "universal" reference with excellent accuracy.

Chitosan-Enclosed Menadione Sodium Bisulfite as an Environmentally Friendly Alternative to Enhance Biostimulant Properties against Drought.

Biostimulants are an interesting strategy to increase crop tolerance to water deficits, and there is an extensive bibliography on them. However, most of them need to be treated continuously to increase protection throughout the growth cycle. In this context, we chose menadione sodium bisulfite, whose protective effect against water deficit has been previously demonstrated but only for a short period of time. Nanoencapsulation seems to be an interesting way to improve the properties of biostimulants. Our results show that menadione sodium bisulfite (MSB) encapsulated in chitosan/tripolyphosphate nanoparticles can increase the system's tolerance against an imposed water deficit and delay the need for retreatment by at least 1 week, accelerating plant recovery after rehydration. This highlights the positive properties of nanoencapsulation and shows how a simple encapsulation process can significantly improve the biostimulant protective properties, opening up new possibilities to be explored under field conditions to cope with water-deficit stress.

Abscisic acid mimic-fluorine derivative 4 alleviates water deficit stress by regulating ABA-responsive genes, proline accumulation, CO2 assimilation, water use efficiency and better nutrient uptake in tomato plants.

Water deficit represents a serious limitation for agriculture and both genetic and chemical approaches are being used to cope with this stress and maintain plant yield. Next-generation agrochemicals that control stomatal aperture are promising for controlling water use efficiency. For example, chemical control of abscisic acid (ABA) signaling through ABA-receptor agonists is a powerful method to activate plant adaptation to water deficit. Such agonists are molecules able to bind and activate ABA receptors and, although their development has experienced significant advances in the last decade, few translational studies have been performed in crops. Here, we describe protection by the ABA mimic-fluorine derivative 4 (AMF4) agonist of the vegetative growth in tomato plants subjected to water restriction. Photosynthesis in mock-treated plants is markedly impaired under water deficit conditions, whereas AMF4 treatment notably improves CO2 assimilation, the relative plant water content and growth. As expected for an antitranspirant molecule, AMF4 treatment diminishes stomatal conductance and transpiration in the first phase of the experiment; however, when photosynthesis declines in mock-treated plants as stress persists, higher photosynthetic and transpiration parameters are recorded in agonist-treated plants. Additionally, AMF4 increases proline levels over those achieved in mock-treated plants in response to water deficit. Thus water deficit and AMF4 cooperate to upregulate P5CS1 through both ABA-independent and ABA-dependent pathways, and therefore, higher proline levels are produced Finally, analysis of macronutrients reveals higher levels of Ca, K and Mg in AMF4- compared to mock-treated plants subjected to water deficit. Overall, these physiological analyses reveal a protective effect of AMF4 over photosynthesis under water deficit and enhanced water use efficiency after agonist treatment. In summary, AMF4 treatment is a promising approach for farmers to protect the vegetative growth of tomatoes under water deficit stress.

Biostimulant activity of Galaxaura rugosa seaweed extracts against water deficit stress in tomato seedlings involves activation of ABA signaling.

Water scarcity is a serious constraint for agriculture, and global warming and climate change can exacerbate it in many areas. Therefore, sustainable approaches must be implemented to deal with current and future water scarcity scenarios. Genetic and chemical approaches are being applied to manage this limitation and maintain crop yields. In particular, biostimulants obtained from natural sources such as marine algae are promising aids for coping with water deficit stress in agriculture. Here we present a bioprospection study of extracts of the macroalgae Bonnemaisonia hamifera, Galaxaura rugosa, Dasycladus vermicularis, Ulva clathrata, Cystoseira foeniculacea, Cystoseira humilis, Lobophora dagamae, Colpomenia sinuosa and Halopteris scoparia from the north coast of Tenerife, in the Canary Islands. The aqueous extracts of Bonnemaisonia hamifera, Galaxaura rugosa, Dasycladus vermicularis and Cystoseira humilis show biostimulant activity against water deficit stress in tomato seedlings under controlled conditions, providing higher tolerance than the mock-treated control. The Galaxaura rugosa extract showed the highest biostimulant activity against water deficit stress. We demonstrate that this positive effect involves the activation of the abscisic acid (ABA) pathway in Arabidopsis thaliana (arabidopsis) and Solanum lycopersicum (tomato). Application of G. rugosa extract to the root system by drenching tomato seedlings subjected to water deficit leads to improved CO2 assimilation and water use efficiency (WUEp), compared to mock-treated plants. These results highlight a new potential seaweed source of substances with osmoprotectant properties, useful for biostimulant development. Future studies may provide further insight into which components of the seaweed extract induce activation of the ABA pathway.

The use of chitosan oligosaccharide to improve artemisinin yield in well-watered and drought-stressed plants.

Introduction: Artemisinin is a secondary metabolite well-known for its use in the treatment of malaria. It also displays other antimicrobial activities which further increase its interest. At present, Artemisia annua is the sole commercial source of the substance, and its production is limited, leading to a global deficit in supply. Furthermore, the cultivation of A. annua is being threatened by climate change. Specifically, drought stress is a major concern for plant development and productivity, but, on the other hand, moderate stress levels can elicit the production of secondary metabolites, with a putative synergistic interaction with elicitors such as chitosan oligosaccharides (COS). Therefore, the development of strategies to increase yield has prompted much interest. With this aim, the effects on artemisinin production under drought stress and treatment with COS, as well as physiological changes in A. annua plants are presented in this study. Methods: Plants were separated into two groups, well-watered (WW) and drought-stressed (DS) plants, and in each group, four concentrations of COS were applied (0, 50,100 and 200 mg•L-1). Afterwards, water stress was imposed by withholding irrigation for 9 days. Results: Therefore, when A. annua was well watered, COS did not improve plant growth, and the upregulation of antioxidant enzymes hindered the production of artemisinin. On the other hand, during drought stress, COS treatment did not alleviate the decline in growth at any concentration tested. However, higher doses improved the water status since leaf water potential (YL) improved by 50.64% and relative water content (RWC) by 33.84% compared to DS plants without COS treatment. Moreover, the combination of COS and drought stress caused damage to the plant's antioxidant enzyme defence, particularly APX and GR, and reduced the amount of phenols and flavonoids. This resulted in increased ROS production and enhanced artemisinin content by 34.40% in DS plants treated with 200 mg•L-1 COS, compared to control plants. Conclusion: These findings underscore the critical role of ROS in artemisinin biosynthesis and suggest that COS treatment may boost artemisinin yield in crop production, even under drought conditions.

Addressing the contribution of small molecule-based biostimulants to the biofortification of maize in a water restriction scenario.

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.

Root treatment with a vitamin K3 derivative: a promising alternative to synthetic fungicides against Botrytis cinerea in tomato plants.

BACKGROUND: Botrytis cinerea, the causal agent of gray mold has a great economic impact on several important crops. This necrotrophic fungus causes disease symptoms during vegetative growth and also into postharvest stages. The current method to combat this disease is fungicide application, with high economic costs and environmentally unsustainable impacts. Moreover, there is an increasing general public health concern about these strategies of crop protection. We studied the protection of tomato plants against B. cinerea by previous root treatment with menadione sodium bisulfite (MSB), a known plant defense activator. RESULTS: Root treatment 48 h before inoculation with MSB 0.6 mmol L-1 reduced leaf lesion diameter by 30% and notably cell deaths, compared to control plants 72 h after inoculation. We studied the expression level of several pathogenesis-related (PR) genes from different defense transduction pathways, and found that MSB primes higher PR1 expression against B. cinerea. However, this stronger induced resistance was impaired in transgenic salicylic acid-deficient NahG line. Additionally, in the absence of pathogen challenge, MSB increased tomato plant growth by 28% after 10 days. Our data provide evidence that MSB protects tomato plants against B. cinerea by priming defense responses through the salicylic acid (SA)-dependent signaling pathway and reducing oxidative stress. CONCLUSION: This work confirms the efficacy of MSB as plant defense activator against B. cinerea and presents a novel alternative to combat gray mold in important crops.

Encapsulation with Natural Polymers to Improve the Properties of Biostimulants in Agriculture.

Encapsulation in agriculture today is practically focused on agrochemicals such as pesticides, herbicides, fungicides, or fertilizers to enhance the protective or nutritive aspects of the entrapped active ingredients. However, one of the most promising and environmentally friendly technologies, biostimulants, is hardly explored in this field. Encapsulation of biostimulants could indeed be an excellent means of counteracting the problems posed by their nature: they are easily biodegradable, and most of them run off through the soil, losing most of the compounds, thus becoming inaccessible to plants. In this respect, encapsulation seems to be a practical and profitable way to increase the stability and durability of biostimulants under field conditions. This review paper aims to provide researchers working on plant biostimulants with a quick overview of how to get started with encapsulation. Here we describe different techniques and offer protocols and suggestions for introduction to polymer science to improve the properties of biostimulants for future agricultural applications.

A Beginner's Guide to Osmoprotection by Biostimulants.

Water is indispensable for the life of any organism on Earth. Consequently, osmotic stress due to salinity and drought is the greatest threat to crop productivity. Ongoing climate change includes rising temperatures and less precipitation over large areas of the planet. This is leading to increased vulnerability to the drought conditions that habitually threaten food security in many countries. Such a scenario poses a daunting challenge for scientists: the search for innovative solutions to save water and cultivate under water deficit. A search for formulations including biostimulants capable of improving tolerance to this stress is a promising specific approach. This review updates the most recent state of the art in the field.

Pure Organic Active Compounds Against Abiotic Stress: A Biostimulant Overview.

Biostimulants (BSs) are probably one of the most promising alternatives nowadays to cope with yield losses caused by plant stress, which are intensified by climate change. Biostimulants comprise many different compounds with positive effects on plants, excluding pesticides and chemical fertilisers. Usually mixtures such as lixiviates from proteins or algal extracts have been used, but currently companies are interested in more specific compounds that are capable of increasing tolerance against abiotic stress. Individual application of a pure active compound offers researchers the opportunity to better standarise formulations, learn more about the plant defence process itself and assist the agrochemical industry in the development of new products. This review attempts to summarise the state of the art regarding various families of organic compounds and their mode/mechanism of action as BSs, and how they can help maximise agricultural yields under stress conditions aggravated by climate change.

Molecular analysis of menadione-induced resistance against biotic stress in Arabidopsis.

Menadione sodium bisulphite (MSB) is a water-soluble derivative of vitamin K3, or menadione, and has been previously demonstrated to function as a plant defence activator against several pathogens in several plant species. However, there are no reports of the role of this vitamin in the induction of resistance in the plant model Arabidopsis thaliana. In the current study, we demonstrate that MSB induces resistance by priming in Arabidopsis against the virulent strain Pseudomonas syringae pv. tomato DC3000 (Pto) without inducing necrosis or visible damage. Changes in gene expression in response to 0.2 mm MSB were analysed in Arabidopsis at 3, 6 and 24 h post-treatment using microarray technology. In general, the treatment with MSB does not correlate with other publicly available data, thus MSB produces a unique molecular footprint. We observed 158 differentially regulated genes among all the possible trends. More up-regulated genes are included in categories such as 'response to stress' than the background, and the behaviour of these genes in different treatments confirms their role in response to biotic and abiotic stress. In addition, there is an over-representation of the G-box in their promoters. Some interesting functions are represented among the individual up-regulated genes, such as glutathione S-transferases, transcription factors (including putative regulators of the G-box) and cytochrome P450s. This work provides a wide insight into the molecular cues underlying the effect of MSB as a plant resistance inducer.

Menadione Sodium Bisulphite (MSB) enhances the resistance response of tomato, leading to repel mollusc pests.

BACKGROUND: Snails and slugs are terrestrial gastropods representing an important biotic stress that adversely affects crop yields. These pests are typically controlled with molluscicides, which produce pollution and toxicity and further induce the evolution of resistance mechanisms, making pest management even more challenging. In our work, we have assessed the efficacy of two different plant defence activators, menadione sodium bisulphite (MSB) and 1,2,3-benzothiadiazole-7-thiocarboxylic acid S-methyl ester (BTH), as inducers of resistance mechanisms of the model plant for defence, Solanum lycopersicum, against the generalist mollusc Theba grasseti (Helicidae). The study was designed to test the feeding behaviour and choice of snails, and also to analyse the expression profile of different genes specifically involved in defence against herbivores and wounds. RESULTS: Our data suggest that, through the downregulation of the terpene volatile genes and the production of proteinase inhibitors, treated MSB plants may be less apparent to herbivores that use herbivore-induced plant volatiles for host location. By contrast, BTH was not effective in the treatment of the pest, probably owing to an antagonistic effect derived from the induction of both salicylic-acid-dependent and jasmonic-acid-dependent pathways. CONCLUSIONS: This information is crucial to determine the genetic basis of the choice of terrestrial gastropod herbivores in tomato, providing valuable insight into how the plant defence activators could control herbivore pests in plants. Our work not only reports for the first time the interaction between tomato and a mollusc pest but also presents the action of two plant defence inductors that seems to produce opposed responses by inducing resistance mechanisms through different defence pathways.