Plant Extracellular Vesicles: Current Landscape and Future Directions.

Plant cells secrete membrane-enclosed micrometer- and nanometer-sized vesicles that, similarly to the extracellular vesicles (EVs) released by mammalian or bacterial cells, carry a complex molecular cargo of proteins, nucleic acids, lipids, and primary and secondary metabolites. While it is technically complicated to isolate EVs from whole plants or their tissues, in vitro plant cell cultures provide excellent model systems for their study. Plant EVs have been isolated from the conditioned culture media of plant cell, pollen, hairy root, and protoplast cultures, and recent studies have gathered important structural and biological data that provide a framework to decipher their physiological roles and unveil previously unacknowledged links to their diverse biological functions. The primary function of plant EVs seems to be in the secretion that underlies cell growth and morphogenesis, cell wall composition, and cell-cell communication processes. Besides their physiological functions, plant EVs may participate in defence mechanisms against different plant pathogens, including fungi, viruses, and bacteria. Whereas edible and medicinal-plant-derived nanovesicles isolated from homogenised plant materials ex vivo are widely studied and exploited, today, plant EV research is still in its infancy. This review, for the first time, highlights the different in vitro sources that have been used to isolate plant EVs, together with the structural and biological studies that investigate the molecular cargo, and pinpoints the possible role of plant EVs as mediators in plant-pathogen interactions, which may contribute to opening up new scenarios for agricultural applications, biotechnology, and innovative strategies for plant disease management.

Plant and Microalgae Derived Peptides Are Advantageously Employed as Bioactive Compounds in Cosmetics.

Bioactive peptides (BP) are specific protein fragments that are physiologically important for most living organisms. It is proven that in humans they are involved in a wide range of therapeutic activities as antihypertensive, antioxidant, anti-tumoral, anti-proliferative, hypocholesterolemic, and anti-inflammatory. In plants, BP are involved in the defense response, as well as in the cellular signaling and the development regulation. Most of the peptides used as ingredients in health-promoting foods, dietary supplements, pharmaceutical, and cosmeceutical preparations are obtained by chemical synthesis or by partial digestion of animal proteins. This makes them not fully accepted by the consumers because of the risks associated with solvent contamination or the use of animal derived substances. On the other hand, plant and microalgae derived peptides are known to be selective, effective, safe, and well tolerated once consumed, thus they have got a great potential for use in functional foods, drugs, and cosmetic products. In fact, the interest in the plant and microalgae derived BP is rapidly increasing and in this review, we highlight and discuss the current knowledge about their studies and applications in the cosmetic field.

The Growth Differentiation Factor 11 is Involved in Skin Fibroblast Ageing and is Induced by a Preparation of Peptides and Sugars Derived from Plant Cell Cultures.

Ageing is a complex and progressive phenomenon, during which the accumulation of morphological and chemical changes seriously compromises the capacity of the cells to proliferate and fulfil their biological tasks. The increase in the average age of the world population, associated with a higher occurrence of age-related diseases, is prompting scientific research to look for new strategies and molecular targets that may help in alleviating age-related phenotypes. Growth factors, responsible for modulating several aging markers in many tissues and organs, represent valuable targets to fight age-associated dysfunctions. The growth differentiation factor GDF11, a TGF-ß family member, has been associated with the maintenance of youth phenotypes in different human tissues and organs, and in the skin has been related to an inhibition of the inflammatory response. We investigated the role of GDF11 in skin dermal fibroblasts, and we observed that its expression and activity were reduced in fibroblasts deriving from adult donors compared to neonatal ones. The main effect of GDF11 was the induction of collagen I and III, in both neonatal and adult fibroblasts, by triggering Smad signalling in a TGF-ß-like fashion. Moreover, by analysing a number of plant extracts having GDF11 inducing activity, we found that a peptide/sugar preparation, obtained from Lotus japonicus somatic embryo cultures, was capable of restoring GDF11 expression in older fibroblasts and to activate the synthesis of collagen I, collagen III and periostin, an important protein involved in collagen assembly.

The Hydrophobin HYTLO1 Secreted by the Biocontrol Fungus Trichoderma longibrachiatum Triggers a NAADP-Mediated Calcium Signalling Pathway in Lotus japonicus.

Trichoderma filamentous fungi are increasingly used as biocontrol agents and plant biostimulants. Growing evidence indicates that part of the beneficial effects is mediated by the activity of fungal metabolites on the plant host. We have investigated the mechanism of plant perception of HYTLO1, a hydrophobin abundantly secreted by Trichoderma longibrachiatum, which may play an important role in the early stages of the plant-fungus interaction. Aequorin-expressing Lotus japonicus suspension cell cultures responded to HYTLO1 with a rapid cytosolic Ca2+ increase that dissipated within 30 min, followed by the activation of the defence-related genes MPK3, WRK33, and CP450. The Ca2+-dependence of these gene expression was demonstrated by using the extracellular Ca2+ chelator EGTA and Ned-19, a potent inhibitor of the nicotinic acid adenine dinucleotide phosphate (NAADP) receptor in animal cells, which effectively blocked the HYTLO1-induced Ca2+ elevation. Immunocytochemical analyses showed the localization of the fungal hydrophobin at the plant cell surface, where it forms a protein film covering the plant cell wall. Our data demonstrate the Ca2+-mediated perception by plant cells of a key metabolite secreted by a biocontrol fungus, and provide the first evidence of the involvement of NAADP-gated Ca2+ release in a signalling pathway triggered by a biotic stimulus.

Brassica rapa hairy root extracts promote skin depigmentation by modulating melanin production and distribution.

BACKGROUND: Skin whitening products, used for ages by Asian people for cultural and esthetic purposes, are very popular nowadays in Western countries as well, where the need to inhibit skin spots after sun exposure has become not only a cosmetic but also a health-related issue. Thus, the development of effective and safe depigmenting agents derived from natural products gets continuous attention by cosmetic brands and consumers. OBJECTIVES: The aim of this study was to determine the effects of two preparations, obtained from the hairy root cultures of the species Brassica rapa, on melanogenesis and the expression of the extracellular matrix proteins involved in a correct pigment distribution. METHODS: The two preparations, obtained by water-ethanol extraction and by digestion of cell-wall glycoproteins of the root cells, were chemically characterized and tested on skin cell cultures and on human skin explants to investigate on their dermatological activities. RESULTS: Both the extracts were able to decrease melanin synthesis pathway in melanocytes and modulate the expression of genes involved in melanin distribution. One of the extracts was also effective in inducing the expression of laminin-5 and collagen IV, involved into the maintenance of tissue integrity. The two extracts, when tested together on human skin explants, demonstrated a good synergic hypopigmenting activity. CONCLUSIONS: Taken together, the results indicate that the extracts from B. rapa root cultures can be employed as cosmetic active ingredients in skin whitening products and as potential therapeutic agents for treating pigmentation disorders.

PII Overexpression in Lotus japonicus Affects Nodule Activity in Permissive Low-Nitrogen Conditions and Increases Nodule Numbers in High Nitrogen Treated Plants.

We report here the first characterization of a GLNB1 gene coding for the PII protein in leguminous plants. The main purpose of this work was the investigation of the possible roles played by this multifunctional protein in nodulation pathways. The Lotus japonicus LjGLB1 gene shows a significant transcriptional regulation during the light-dark cycle and different nitrogen availability, conditions that strongly affect nodule formation, development, and functioning. We also report analysis of the spatial profile of expression of LjGLB1 in root and nodule tissues and of the protein's subcellular localization. Transgenic L. japonicus lines overexpressing the PII protein were obtained and tested for the analysis of the symbiotic responses in different conditions. The uncoupling of PII from its native regulation affects nitrogenase activity and nodule polyamine content. Furthermore, our results suggest the involvement of PII in the signaling of the nitrogen nutritional status affecting the legumes' predisposition for nodule formation.

A tomato stem cell extract, containing antioxidant compounds and metal chelating factors, protects skin cells from heavy metal-induced damages.

Heavy metals can cause several genotoxic effects on cells, including oxidative stress, DNA sequence breakage and protein modification. Among the body organs, skin is certainly the most exposed to heavy metal stress and thus the most damaged by the toxic effects that these chemicals cause. Moreover, heavy metals, in particular nickel, can induce the over-expression of collagenases (enzymes responsible for collagen degradation), leading to weakening of the skin extracellular matrix. Plants have evolved sophisticated mechanisms to protect their cells from heavy metal toxicity, including the synthesis of metal chelating proteins and peptides, such as metallothioneins and phytochelatins (PC), which capture the metals and prevent the damages on the cellular structures. To protect human skin cells from heavy metal toxicity, we developed a new cosmetic active ingredient from Lycopersicon esculentum (tomato) cultured stem cells. This product, besides its high content of antioxidant compounds, contained PC, effective in the protection of skin cells towards heavy metal toxicity. We have demonstrated that this new product preserves nuclear DNA integrity from heavy metal damages, by inducing genes responsible for DNA repair and protection, and neutralizes the effect of heavy metals on collagen degradation, by inhibiting collagenase expression and inducing the synthesis of new collagen.

Characterization of a developmental root response caused by external ammonium supply in Lotus japonicus.

Plants respond to changes of nutrient availability in the soil by modulating their root system developmental plan. This response is mediated by systemic changes of the nutritional status and/or by local perception of specific signals. The effect of nitrate on Arabidopsis (Arabidopsis thaliana) root development represents a paradigm of these responses, and nitrate transporters are involved both in local and systemic control. Ammonium (NH(4)(+)) represents an important nitrogen (N) source for plants, although toxicity symptoms are often associated with high NH(4)(+) concentration when this is present as the only N source. The reason for these effects is still controversial, and mechanisms associating ammonium supply and plant developmental programs are completely unknown. We determined in Lotus japonicus the range of ammonium concentration that significantly inhibits the elongation of primary and lateral roots without affecting the biomass of the shoot. The comparison of the growth phenotypes in different N conditions indicated the specificity of the ammonium effect, suggesting that this was not mediated by assimilatory negative feedback mechanisms. In the range of inhibitory NH(4)(+) conditions, only the LjAMT1;3 gene, among the members of the LjAMT1 family, showed a strong increased transcription that was reflected by an enlarged topology of expression. Remarkably, the short-root phenotype was phenocopied in transgenic lines by LjAMT1;3 overexpression independently of ammonium supply, and the same phenotype was not induced by another AMT1 member. These data describe a new plant mechanism to cope with environmental changes, giving preliminary information on putative actors involved in this specific ammonium-induced response.

Tissue-specific down-regulation of LjAMT1;1 compromises nodule function and enhances nodulation in Lotus japonicus.

Plant ammonium transporters of the AMT1 family are involved in N-uptake from the soil and ammonium transport, and recycling within the plant. Although AMT1 genes are known to be expressed in nitrogen-fixing nodules of legumes, their precise roles in this specialized organ remain unknown. We have taken a reverse-genetic approach to decipher the physiological role of LjAMT1;1 in Lotus japonicus nodules. LjAMT1;1 is normally expressed in both the infected zone and the vascular tissue of Lotus nodules. Inhibition of LjAMT1;1 gene expression, using an antisense gene construct driven by a leghemoglobin promoter resulted in a substantial reduction of LjAMT1;1 transcript in the infected tissue but not the vascular bundles of transgenic plants. As a result, the nitrogen-fixing activity of nodules was partially impaired and nodule number increased compared to control plants. Expression of LjAMT1;1-GFP fusion protein in plant cells indicated a plasma-membrane location for the LjAMT1;1 protein. Taken together, the results are consistent with a role of LjAMT1;1 in retaining ammonium derived from symbiotic nitrogen fixation in plant cells prior to its assimilation.

Differential effects of combined N sources on early steps of the Nod factor-dependent transduction pathway in Lotus japonicus.

The development of nitrogen-fixing nodules in legumes is induced by perception of lipochitin-oligosaccharide signals secreted by a bacterial symbiont. Nitrogen (N) starvation is a prerequisite for the formation, development, and function of root nodules, and high levels of combined N in the form of nitrate or ammonium can completely abolish nodule formation. We distinguished between nitrate and ammonium inhibitory effects by identifying when and where these combined N sources interfere with the Nod-factor-induced pathway. Furthermore, we present a small-scale analysis of the expression profile, under different N conditions, of recently identified genes involved in the Nod-factor-induced pathway. In the presence of high levels of nitrate or ammonium, the NIN gene fails to be induced 24 h after the addition of Nod factor compared with plants grown under N-free conditions. This induction is restored in the hypernodulating nitrate-tolerant har1-3 mutant only in the presence of 10 and 20 mM KNO3. These results were confirmed in Lotus plants inoculated with Mesorhizobium loti. NIN plays a key role in the nodule organogenesis program and its downregulation may represent a crucial event in the nitrate-dependent pathway leading to the inhibition of nodule organogenesis.

Improved procedures for in vitro regeneration and for phenotypic analysis in the model legume Lotus japonicus.

As a prerequisite for the development of an efficient gene transfer methodology, the possibility of inducing direct somatic embryogenesis in Lotus japonicus (Regel) K. Larsen explants was investigated. Petiole bases, cotyledons, hypocotyls and stem segments were cultivated in the presence of different amounts of benzylaminopurine (BAP) and / or thidiazuron (TDZ). Regeneration was achieved differentially in the different explants and a higher efficiency of shoot formation was obtained with TDZ. By maintaining the same TDZ regime a second cycle of morphogenesis was achieved and the histological analysis of these structures indicated unambiguously their somatic embryogenic nature. Thidiazuron was also tested as an agent to improve the kinetics of shoot formation in a Lotus japonicus transformation-regeneration procedure based on indirect organogenesis. A very significant, highly reproducible, increase in the rate of the shoot formation was observed in independent transformation experiments. We also present an extensive analysis of the feasibility and reproducibility of an in vitro procedure, which can be very useful for the screening of symbiotic phenotypes in transgenic Lotus plants and for the analysis of the cascade of molecular and cytological events occurring soon after Mesorhizobium loti infection.

Characterization of three functional high-affinity ammonium transporters in Lotus japonicus with differential transcriptional regulation and spatial expression.

Ammonium is a primary source of nitrogen for plants. In legume plants ammonium can also be obtained by symbiotic nitrogen fixation, and NH(4)(+) is also a regulator of early and late symbiotic interaction steps. Ammonium transporters are likely to play important roles in the control of nodule formation as well as in nitrogen assimilation. Two new genes, LjAMT1;2 and LjAMT1;3, were cloned from Lotus japonicus. Both were able to complement the growth defect of a yeast (Saccharomyces cerevisiae) ammonium transport mutant. Measurement of [(14)C]methylammonium uptake rates and competition experiments revealed that each transporter had a high affinity for NH(4)(+). The K(i) for ammonium was 1.7, 3, and 15 microm for LjAMT1;1, 1;2, and 1;3, respectively. Real-time PCR revealed higher expression of LjAMT1;1, 1;2, and 1;3 genes in leaves than in roots and nodule, with expression levels decreasing in the order LjAMT1;1 > 1;2 > 1;3 except in flowers, in which LjAMT1;3 was expressed at higher level than in leaves, and LjAMT1;1 showed the lowest level of expression. Expression of LjAMT1;1 and 1;2 in roots was induced by nitrogen deprivation. Expression of LjAMT1;1 was repressed in leaves exposed to elevated CO(2) concentrations, which also suppress photorespiration. Tissue and cellular localization of LjAMT1 genes expression, using promoter-beta-glucuronidase and in situ RNA hybridization approaches, revealed distinct cellular spatial localization in different organs, including nodules, suggesting differential roles in the nitrogen metabolism of these organs.