Brassica oleracea L. var. italica Aquaporin Reconstituted Proteoliposomes as Nanosystems for Resveratrol Encapsulation.

Aquaporins (AQPs), membrane proteins responsible for facilitating water transport, found in plant membrane vesicles (MV), have been related to the functionality and stability of MV. We focused on AQPs obtained from broccoli, as they show potential for biotechnological applications. To gain further insight into the role of AQPs in MV, we describe the heterologous overexpression of two broccoli AQPs (BoPIP1;2 and BoPIP2;2) in Pichia pastoris, resulting in their purification with high yield (0.14 and 0.99 mg per gram cells for BoPIP1;2 and BoPIP2;2). We reconstituted AQPs in liposomes to study their functionality, and the size of proteoliposomes did not change concerning liposomes. BoPIP2;2 facilitated water transport, which was preserved for seven days at 4 °C and at room temperature but not at 37 °C. BoPIP2;2 was incorporated into liposomes to encapsulate a resveratrol extract, resulting in increased entrapment efficiency (EE) compared to conventional liposomes. Molecular docking was utilized to identify binding sites in PIP2s for resveratrol, highlighting the role of aquaporins in the improved EE. Moreover, interactions between plant AQP and human integrin were shown, which may increase internalization by the human target cells. Our results suggest AQP-based alternative encapsulation systems can be used in specifically targeted biotechnological applications.

Exploring Phenolic Compounds in Crop By-Products for Cosmetic Efficacy.

Phenolic compounds represent a group of secondary metabolites that serve essential functions in plants. Beyond their positive impact on plants, these phenolic metabolites, often referred to as polyphenols, possess a range of biological properties that can promote skin health. Scientific research indicates that topically using phenolics derived from plants can be advantageous, but their activity and stability highly depend on storage of the source material and the extraction method. These compounds have the ability to relieve symptoms and hinder the progression of different skin diseases. Because they come from natural sources and have minimal toxicity, phenolic compounds show potential in addressing the causes and effects of skin aging, skin diseases, and various types of skin damage, such as wounds and burns. Hence, this review provides extensive information on the particular crops from which by-product phenolic compounds can be sourced, also emphasizing the need to conduct research according to proper plant material storage practices and the choice of the best extracting method, along with an examination of their specific functions and the mechanisms by which they act to protect skin.

Exploring membrane vesicles in citrus fruits: a comparative analysis of conventional and organic farming approaches.

BACKGROUND: Recently, vesicles derived from plant cell membranes have received attention for their potential use as active biomolecules and nanocarriers, and obtaining them from organic crops may be an interesting option because different farming systems can affect production, plant secondary metabolism and biochemistry of cell membranes. The present study aimed to determine how organic and conventional farming affects the mineral nutrition, gas exchange, CO2 fixation and biochemical composition of lemon fruits, which could have an impact on the different fractions of cell membranes in pulp and juice. RESULTS: Organic trees had higher intrinsic water use efficiency (WUEi) but conventional trees had higher stomatal conductance (gs) and nitrogen use efficiency (NUtE). Also, organic lemons had significantly higher levels of some micronutrients (Ca, Cu, Fe and Zn). Second, the main differences in the membrane vesicles showed that organic pulp vesicles had a higher antioxidant activity and more oleic acid, whereas both types of vesicles from conventional lemons had more linoleic acid. CONCLUSION: In conclusion, organic farming did not alter carbon fixation parameters but impacted nitrogen fixation and water uptake, and resulted in higher micronutrient levels in lemons. These mineral nutritional changes could be related to the higher production of membranes that showed suitable morphological traits and a high antioxidant activity, positively correlated with a high amount of oleic acid, which could have stronger cell protection characteristics. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Confronting Secondary Metabolites with Water Uptake and Transport in Plants under Abiotic Stress.

Phenolic compounds and glucosinolates are secondary plant metabolites that play fundamental roles in plant resistance to abiotic stress. These compounds have been found to increase in stress situations related to plant adaptive capacity. This review assesses the functions of phenolic compounds and glucosinolates in plant interactions involving abiotic stresses such as drought, salinity, high temperature, metals toxicity, and mineral deficiency or excess. Furthermore, their relation with water uptake and transport mediated through aquaporins is reviewed. In this way, the increases of phenolic compounds and glucosinolate synthesis have been related to primary responses to abiotic stress and induction of resistance. Thus, their metabolic pathways, root exudation, and external application are related to internal cell and tissue movement, with a lack of information in this latter aspect.

Membrane Vesicles for Nanoencapsulated Sulforaphane Increased Their Anti-Inflammatory Role on an In Vitro Human Macrophage Model.

At present, there is a growing interest in finding new non-toxic anti-inflammatory drugs to treat inflammation, which is a key pathology in the development of several diseases with considerable mortality. Sulforaphane (SFN), a bioactive compound derived from Brassica plants, was shown to be promising due to its anti-inflammatory properties and great potential, though its actual clinical use is limited due to its poor stability and bioavailability. In this sense, the use of nanocarriers could solve stability-related problems. In the current study, sulforaphane loaded into membrane vesicles derived from broccoli plants was studied to determine the anti-inflammatory potential in a human-macrophage-like in vitro cell model under both normal and inflammatory conditions. On the one hand, the release of SFN from membrane vesicles was modeled in vitro, and two release phases were stabilized, one faster and the other slower due to the interaction between SFN and membrane proteins, such as aquaporins. Furthermore, the anti-inflammatory action of sulforaphane-loaded membrane vesicles was demonstrated, as a decrease in interleukins crucial for the development of inflammation, such as TNF-α, IL-1ß and IL-6, was observed. Furthermore, these results also showed that membrane vesicles by themselves had anti-inflammatory properties, opening the possibility of new lines of research to study these vesicles, not only as carriers but also as active compounds.

Potential of Sulforaphane and Broccoli Membrane Vesicles as Regulators of M1/M2 Human Macrophage Activity.

Macrophages have emerged as important therapeutic targets in many human diseases. The aim of this study was to analyze the effect of broccoli membrane vesicles and sulphoraphane (SFN), either free or encapsulated, on the activity of human monocyte-derived M1 and M2 macrophage primary culture. Our results show that exposure for 24 h to SFN 25 µM, free and encapsulated, induced a potent reduction on the activity of human M1 and M2 macrophages, downregulating proinflammatory and anti-inflammatory cytokines and phagocytic capability on C. albicans. The broccoli membrane vesicles do not represent inert nanocarriers, as they have low amounts of bioactive compounds, being able to modulate the cytokine production, depending on the inflammatory state of the cells. They could induce opposite effects to that of higher doses of SFN, reflecting its hormetic effect. These data reinforce the potential use of broccoli compounds as therapeutic agents not only for inflammatory diseases, but they also open new clinical possibilities for applications in other diseases related to immunodeficiency, autoimmunity, or in cancer therapy. Considering the variability of their biological effects in different scenarios, a proper therapeutic strategy with Brassica bioactive compounds should be designed for each pathology.

Bioactive peptides from broccoli stems strongly enhance regenerative keratinocytes by stimulating controlled proliferation.

CONTEXT: As the interest on the research of plant derived bioactive peptides (BPs) for nutraceutical, cosmeceutical and medical applications is increasing, in this work, the application of peptide derived from broccoli to keratinocytes was studied. OBJECTIVE: We focussed on the characterization of different peptides hydrolysates from broccoli stems [extracted from total protein (E) and from membrane protein (MF)], and their activity when applied to human keratinocytes. MATERIALS AND METHODS: Peptide mixtures from broccoli stems (E and MF) were characterized by proteomics. They were applied to HaCaT cells in order to study cytotoxicity in a concentration range between 20 and 0.15625 µg of protein/mL and wound healing was studied after 24 and 48 h of treatment application. Also, proteomic and gene expression of keratinocytes were analysed. RESULTS: Depending on the source, proteins varied in peptide and amino acid composition. An increased proliferation of keratinocytes was shown after the application of the E peptides mixtures, reaching 190% with the lowest concentrations, but enhanced wound healing repair with E and MF appeared, reaching 59% of wound closure after 48 h. At the gene expression and protein levels of keratinocytes, the upregulation of anti-oncogene p53 and keratinization factors were observed. DISCUSSION: These results suggest that peptide mixtures obtained from broccoli augmented cell proliferation and prevented the carcinogenic, uncontrolled growth of the cells, with different mechanisms depending on the protein source. CONCLUSIONS: The results encourage the opening of new lines of research involving the use of Brassica peptides for pharmaceutic or cosmetic use.

Interrelations of nutrient and water transporters in plants under abiotic stress.

Environmental changes cause abiotic stress in plants, primarily through alterations in the uptake of the nutrients and water they require for their metabolism and growth and to maintain their cellular homeostasis. The plasma membranes of cells contain transporter proteins, encoded by their specific genes, responsible for the uptake of nutrients and water (aquaporins). However, their interregulation has rarely been taken into account. Therefore, in this review we identify how the plant genome responds to abiotic stresses such as nutrient deficiency, drought, salinity and low temperature, in relation to both nutrient transporters and aquaporins. Some general responses or regulation mechanisms can be observed under each abiotic stress such as the induction of plasma membrane transporter expression during macronutrient deficiency, the induction of tonoplast transporters and reduction of aquaporins during micronutrients deficiency. However, drought, salinity and low temperatures generally cause an increase in expression of nutrient transporters and aquaporins in tolerant plants. We propose that both types of transporters (nutrients and water) should be considered jointly in order to better understand plant tolerance of stresses.

Nanoencapsulation of sulforaphane in broccoli membrane vesicles and their in vitro antiproliferative activity.

CONTEXT: The development of nanocarriers of plant origin, such as plant cell membranes, has recently been investigated. Also, plant bioactive compounds as sulforaphane (SFN) from broccoli have recognized antioxidant or anticancer properties. OBJECTIVE: To investigate the capacity of membrane vesicles from broccoli (BM-vesicles) to encapsulate SFN and their application in the cancer cell line. MATERIALS AND METHODS: Physicochemical analysis was carried out to characterize BM-vesicles through different approaches: dynamic light scattering, transmission electron microscopy, stopped-flow analysis, and proteomic analysis. They were applied at different concentrations (BM-vesicles at 0.04-0.00315% of protein and SFN at 5, 25, and 100 µM) in SK-MEL-28 cells during 24 h for studying cytotoxicity and gene expression. RESULTS: The entrapment efficiency was 41%. The anticancer activity tested in cells showed a decrease in proliferation when SFN in BM-vesicles was utilized. Expression patterns when SFN was applied in an encapsulated form showed a reduction of cancer markers and an increase of AQP3. Also, the metabolism of SFN occurred inside of cells, and higher SFN penetrated when it was encapsulated. DISCUSSION: The results showed that encapsulated SFN was better absorbed by melanoma cells providing metabolism products and a reduction of cancer molecular markers. Also aquaporin, AQP3 was pointed to as an important marker since it appeared to play a key role in homeostasis due to the importance of water transport in biological processes. CONCLUSION: These results indicate that SFN and SFN encapsulated in BM-vesicles have a high activity for the inhibition of melanocyte development. Therefore, BM-vesicles could serve as nanocarriers for drugs.

Detergent Resistant Membrane Domains in Broccoli Plasma Membrane Associated to the Response to Salinity Stress.

Detergent-resistant membranes (DRMs) microdomains, or "raft lipids", are key components of the plasma membrane (PM), being involved in membrane trafficking, signal transduction, cell wall metabolism or endocytosis. Proteins imbibed in these domains play important roles in these cellular functions, but there are few studies concerning DRMs under abiotic stress. In this work, we determine DRMs from the PM of broccoli roots, the lipid and protein content, the vesicles structure, their water osmotic permeability and a proteomic characterization focused mainly in aquaporin isoforms under salinity (80 mM NaCl). Based on biochemical lipid composition, higher fatty acid saturation and enriched sterol content under stress resulted in membranes, which decreased osmotic water permeability with regard to other PM vesicles, but this permeability was maintained under control and saline conditions; this maintenance may be related to a lower amount of total PIP1 and PIP2. Selective aquaporin isoforms related to the stress response such as PIP1;2 and PIP2;7 were found in DRMs and this protein partitioning may act as a mechanism to regulate aquaporins involved in the response to salt stress. Other proteins related to protein synthesis, metabolism and energy were identified in DRMs independently of the treatment, indicating their preference to organize in DMRs.

Brassica Bioactives Could Ameliorate the Chronic Inflammatory Condition of Endometriosis.

Endometriosis is a chronic, inflammatory, hormone-dependent disease characterized by histological lesions produced by the presence of endometrial tissue outside the uterine cavity. Despite the fact that an estimated 176 million women are affected worldwide by this gynecological disorder, risk factors that cause endometriosis have not been properly defined and current treatments are not efficient. Although the interaction between diet and human health has been the focus of many studies, little information about the correlation of foods and their bioactive derivates with endometriosis is available. In this framework, Brassica crops have emerged as potential candidates for ameliorating the chronic inflammatory condition of endometriosis, due to their abundant content of health-promoting compounds such as glucosinolates and their hydrolysis products, isothiocyanates. Several inflammation-related signaling pathways have been included among the known targets of isothiocyanates, but those involving aquaporin water channels have an important role in endometriosis. Therefore, the aim of this review is to highlight the promising effects of the phytochemicals present in Brassica spp. as major candidates for inclusion in a dietary approach aiming to improve the inflammatory condition of women affected with endometriosis. This review points out the potential roles of glucosinolates and isothiocyanates from Brassicas as anti-inflammatory compounds, which might contribute to a reduction in endometriosis symptoms. In view of these promising results, further investigation of the effect of glucosinolates on chronic inflammatory diseases, either as diet coadjuvants or as therapeutic molecules, should be performed. In addition, we highlight the involvement of aquaporins in the maintenance of immune homeostasis. In brief, glucosinolates and the modulation of cellular water by aquaporins could shed light on new approaches to improve the quality of life for women with endometriosis.

Involvement of glucosinolates and phenolics in the promotion of broccoli seedling growth through the modulation of primary and secondary metabolism.

Secondary metabolites play an essential role in plant defense. However, the role of glucosinolates and phenols in brassica crop yield in the context of environmentally friendly agricultural practices has not been established. Our study investigated the effects of a Brassica extract, rich in these metabolites, on the physiology and metabolism of broccoli (Brassica oleracea L. var. italica) seedlings and the subsequent development of the plants in adult stages. The results showed an increase in growth in the extract-treated seedlings, which was associated with an alteration of primary and secondary metabolism. In particular, there was an increase in the levels of amino acids, phenolic compounds and hormones, while the levels of glucosinolates decreased. Lipid peroxidation diminished in treated plants, indicating improved membrane integrity. Treated plants subsequently grown in hydroponically showed increased water use efficiency, transpiration, and internal carbon, which contributed to the improved growth of these plants. Overall, our findings underscore the potential of the glucosinolates and phenols ratio as essential to improve crop growth and stress tolerance, as well as revealed the interest of studying the mechanisms involved in the possible uptake and integration of GSLs by broccoli seedlings after external application.

Deciphering the effect of salinity and boron stress on broccoli plants reveals that membranes phytosterols and PIP aquaporins facilitate stress adaptation.

Abiotic stresses, such as salinity and boron toxicity/deficiency, are prevalent in arid and semi-arid regions where broccoli is largely cultivated. This study aimed to investigate the physiological response of broccoli leaves to these stresses, focusing on parameters such as growth, relative water content, stomatal conductance, and mineral concentration after 15 days of treatment application. The effects of individual and combined stresses of salinity and boron (deficiency and toxicity) were examined. Additionally, the study explored the molecular aspects of PIP aquaporins in relation to their presence in the plasma membrane and their interaction with the lipid environment. The results showed that the combined stress of salinity and boron deficiency resulted in a significant reduction in plant biomass, suggesting a specific adaptation to this stress combination. Changes in stomatal conductance and mineral nutrient levels indicated that the adaptation mechanisms were associated with water and boron concentration in the leaves. The expression patterns of PIP aquaporins varied among the different stress treatments, either individually or in combination. Furthermore, the presence of aquaporins in the plasma membrane and microsomal fraction highlighted the potential regulatory roles of trafficking along with the membrane composition, particularly the concentration of phytosterols. The results underscore the importance of water transport by aquaporins and their interaction with the sterol composition in the membranes, in facilitating salinity-boron stress adaptation mechanisms.

Modification of Gene Expression of Tomato Plants through Foliar Flavonoid Application in Relation to Enhanced Growth.

The exogenous application of phenolic compounds is increasingly recognized as a valuable strategy for promoting growth and mitigating the adverse effects of abiotic stress. However, the biostimulant effect under optimal conditions has not been thoroughly explored. In this study, we investigated the impact of foliar application of flavonoids, specifically CropBioLife (CBL), on tomato plants grown under controlled conditions. Our study focused on determining growth parameters, such as cell size, and assessing the concentration of hormones. Principal component analysis (PCA) from all physiological variables was determined. Additionally, we utilized high-throughput mRNA-sequencing technology and bioinformatic methodologies to robustly analyze the transcriptomes of tomato leaves regulated by flavonoids. The findings revealed that CBL primarily influenced cell enlargement by 60%, leading to increased growth. Furthermore, CBL-treated plants exhibited higher concentrations of the hormone zeatin, but lower concentrations of IAA (changes of 50%). Moreover, RNA-seq analysis indicated that CBL-treated plants required increased mineral transport and water uptake, as evidenced by gene expression patterns. Genes related to pathways such as fatty acid degradation, phenylpropanoid biosynthesis, and ABC transporters showed regulatory mechanisms governing internal flavonoid biosynthesis, transport, and tissue concentration, ultimately resulting in higher flavonoid concentrations in tomato leaves.

Nanoencapsulation of Pomegranate Extract to Increase Stability and Potential Dermatological Protection.

Pomegranate extract (PG-E) has been reported to exert a protective effect on the skin due to its antioxidant activity. Ingredients rich in phenolic compounds are unstable in extract solutions, and, therefore, the use of a suitable nanosystem to encapsulate this type of extract could be necessary in different biotechnological applications. Thus, we investigated the capacity of Brassica oleracea L. (cauliflower) inflorescence vesicles (CI-vesicles) to encapsulate PG-E and determined the stability and the antioxidant capacity of the system over time. In addition, the protective effect against UV radiation and heavy metals in HaCaT cells was also tested. The CI-vesicles had an entrapment efficiency of around 50%, and accelerated stability tests did not show significant changes in the parameters tested. The results for the HaCaT cells showed the non-cytotoxicity of the CI-vesicles containing PG-E and their protection against heavy metals (lead acetate and mercuric chloride) and UV-B radiation through a reduction of oxidative stress. The reduction of the percentage of deleted mtDNA (mtDNA4977, "common deletion") in UV-treated HaCaT cells due to the presence of CI-vesicles containing PG-E indicated the mechanism of protection. Therefore, the effects of CI-vesicles loaded with PG-E against oxidative stress support their utilization as natural cosmeceuticals to protect skin health against external damage from environmental pollution and UV radiation.

Controversial Regulation of Gene Expression and Protein Transduction of Aquaporins under Drought and Salinity Stress.

Enhancement of the passage of water through membranes is one of the main mechanisms via which cells can maintain their homeostasis under stress conditions, and aquaporins are the main participants in this process. However, in the last few years, a number of studies have reported discrepancies between aquaporin messenger RNA (mRNA) expression and the number of aquaporin proteins synthesised in response to abiotic stress. These observations suggest the existence of post-transcriptional mechanisms which regulate plasma membrane intrinsic protein (PIP) trafficking to the plasma membrane. This indicates that the mRNA synthesis of some aquaporins could be modulated by the accumulation of the corresponding encoded protein, in relation to the turnover of the membranes. This aspect is discussed in terms of the results obtained: on the one hand, with isolated vesicles, in which the level of proteins present provides the membranes with important characteristics such as resistance and stability and, on the other, with isolated proteins reconstituted in artificial liposomes as an in vitro method to address the in vivo physiology of the entire plant.

Plant plasma membrane vesicles interaction with keratinocytes reveals their potential as carriers.

During the last few years, membrane vesicles (as exovesicles) have emerged as potential nanocarriers for therapeutic applications. They are receiving attention due to their proteo-lipid nature, size, biocompatibility and biodegradability. In this work, we investigated the potential use of isolated root plasma membrane vesicles from broccoli plants as nanocarriers. For that, the entrapment efficiency and integrity of the vesicles were determined. Also, the delivery of keratinocytes and penetrability through skin were studied. The results show that the broccoli vesicles had high stability, in relation to their proteins, and high entrapment efficiency. Also, the interaction between the vesicles and keratinocytes was proven by the delivery of an encapsulated fluorescent product into cells and by the detection of plant proteins in the keratinocyte plasma membrane, showing the interactions between the membranes of two species of distinct biological kingdoms. Therefore, these results, together with the capacity of brassica vesicles to cross the skin layers, detected by fluorescent penetration, enable us to propose a type of nanocarrier obtained from natural plant membranes for use in transdermal delivery.

The Expanding Role of Vesicles Containing Aquaporins.

In animals and plants, membrane vesicles containing proteins have been defined as key for biological systems involving different processes such as trafficking or intercellular communication. Docking and fusion of vesicles to the plasma membrane occur in living cells in response to different stimuli, such as environmental changes or hormones, and therefore play an important role in cell homeostasis as vehicles for certain proteins or other substances. Because aquaporins enhance the water permeability of membranes, their role as proteins immersed in vesicles formed of natural membranes is a recent topic of study. They regulate numerous physiological processes and could hence serve new biotechnological purposes. Thus, in this review, we have explored the physiological implications of the trafficking of aquaporins, the mechanisms that control their transit, and the proteins that coregulate the migration. In addition, the importance of exosomes containing aquaporins in the cell-to-cell communication processes in animals and plants have been analyzed, together with their potential uses in biomedicine or biotechnology. The properties of aquaporins make them suitable for use as biomarkers of different aquaporin-related diseases when they are included in exosomes. Finally, the fact that these proteins could be immersed in biomimetic membranes opens future perspectives for new biotechnological applications.

Plasma membrane aquaporins mediates vesicle stability in broccoli.

The use of in vitro membrane vesicles is attractive because of possible applications in therapies. Here we aimed to compare the stability and functionality of plasma membrane vesicles extracted from control and salt-treated broccoli. The impact of the amount of aquaporins was related to plasma membrane osmotic water permeability and the stability of protein secondary structure. Here, we describe for first time an increase in plant aquaporins acetylation under high salinity. Higher osmotic water permeability in NaCl vesicles has been related to higher acetylation, upregulation of aquaporins, and a more stable environment to thermal denaturation. Based on our findings, we propose that aquaporins play an important role in vesicle stability.