An Integrated Multilevel Approach Unveils Complex Seed-Nanoparticle Interactions and Their Implications for Seed Priming.

Nanotechnology has the potential to revolutionize agriculture with the introduction of engineered nanomaterials. However, their use is hindered by high cost, marginal knowledge of their interactions with plants, and unpredictable effects related to massive use in crop cultivation. Nanopriming is an innovative seed priming technology able to match economic, agronomic, and environmental needs in agriculture. The present study was focused on unveiling, by a multilevel integrated approach, undisclosed aspects of seed priming mediated by iron oxide magnetic nanoparticles in pepper seeds (Capsicum annuum), one of the most economically important crops worldwide. Inductively coupled plasma atomic emission mass spectrometry and scanning electron microscopy were used to quantify the MNP uptake and assess seed surface changes. Magnetic resonance imaging mapped the distribution of MNPs prevalently in the seed coat. The application of MNPs significantly enhanced the root and vegetative growth of pepper plants, whereas seed priming with equivalent Fe concentrations supplied as FeCl3 did not yield these positive effects. Finally, global gene expression by RNA-sequencing identified more than 2,200 differentially expressed genes, most of them involved in plant developmental processes and defense mechanisms. Collectively, these data provide evidence on the link between structural seed changes and an extensive transcriptional reprogramming, which boosts the plant growth and primes the embryo to cope with environmental challenges that might occur during the subsequent developmental and growth stages.

Plant hairy roots for the production of extracellular vesicles with antitumor bioactivity.

Plant extracellular vesicles (EVs) concentrate and deliver different types of bioactive molecules in human cells and are excellent candidates for a next-generation drug delivery system. However, the lack of standard protocols for plant EV production and the natural variations of their biomolecular cargo pose serious limitation to their use as therapeutics. To overcome these issues, we set up a versatile and standardized procedure to purify plant EVs from hairy root (HR) cultures, a versatile biotechnological system, already successfully employed as source of bioactive molecules with pharmaceutical and nutraceutical relevance. Herewith, we report that HR of Salvia dominica represent an excellent platform for the production of plant EVs. In particular, EVs derived from S. dominica HRs are small round-shaped vesicles carrying typical EV-associated proteins such as cytoskeletal components, chaperon proteins and integral membrane proteins including the tetraspanin TET-7. Interestingly, the HR-derived EVs showed selective and strong pro-apoptotic activity in pancreatic and mammary cancer cells. These results reveal that plant hairy roots may be considered a new promising tool in plant biotechnology for the production of extracellular vesicles for human health.

Plant-Derived Nano and Microvesicles for Human Health and Therapeutic Potential in Nanomedicine.

Plants produce different types of nano and micro-sized vesicles. Observed for the first time in the 60s, plant nano and microvesicles (PDVs) and their biological role have been inexplicably under investigated for a long time. Proteomic and metabolomic approaches revealed that PDVs carry numerous proteins with antifungal and antimicrobial activity, as well as bioactive metabolites with high pharmaceutical interest. PDVs have also been shown to be also involved in the intercellular transfer of small non-coding RNAs such as microRNAs, suggesting fascinating mechanisms of long-distance gene regulation and horizontal transfer of regulatory RNAs and inter-kingdom communications. High loading capacity, intrinsic biological activities, biocompatibility, and easy permeabilization in cell compartments make plant-derived vesicles excellent natural or bioengineered nanotools for biomedical applications. Growing evidence indicates that PDVs may exert anti-inflammatory, anti-oxidant, and anticancer activities in different in vitro and in vivo models. In addition, clinical trials are currently in progress to test the effectiveness of plant EVs in reducing insulin resistance and in preventing side effects of chemotherapy treatments. In this review, we concisely introduce PDVs, discuss shortly their most important biological and physiological roles in plants and provide clues on the use and the bioengineering of plant nano and microvesicles to develop innovative therapeutic tools in nanomedicine, able to encompass the current drawbacks in the delivery systems in nutraceutical and pharmaceutical technology. Finally, we predict that the advent of intense research efforts on PDVs may disclose new frontiers in plant biotechnology applied to nanomedicine.

Plant Roots Release Small Extracellular Vesicles with Antifungal Activity.

Extracellular Vesicles (EVs) play pivotal roles in cell-to-cell and inter-kingdom communication. Despite their relevant biological implications, the existence and role of plant EVs released into the environment has been unexplored. Herein, we purified round-shaped small vesicles (EVs) by differential ultracentrifugation of a sampling solution containing root exudates of hydroponically grown tomato plants. Biophysical analyses, by means of dynamic light scattering, microfluidic resistive pulse sensing and scanning electron microscopy, showed that the size of root-released EVs range in the nanometric scale (50-100 nm). Shot-gun proteomics of tomato EVs identified 179 unique proteins, several of which are known to be involved in plant-microbe interactions. In addition, the application of root-released EVs induced a significant inhibition of spore germination and of germination tube development of the plant pathogens Fusarium oxysporum, Botrytis cinerea and Alternaria alternata. Interestingly, these EVs contain several proteins involved in plant defense, suggesting that they could be new components of the plant innate immune system.

Grapefruit-Derived Micro and Nanovesicles Show Distinct Metabolome Profiles and Anticancer Activities in the A375 Human Melanoma Cell Line.

Fruit juice is one of the most easily accessible resources for the isolation of plant-derived vesicles. Here we found that micro- and nano-sized vesicles (MVs and NVs) from four Citrus species, C. sinensis, C. limon, C. paradisi and C. aurantium, specifically inhibit the proliferation of lung, skin and breast cancer cells, with no substantial effect on the growth of non-cancer cells. Cellular and molecular analyses demonstrate that grapefruit-derived vesicles cause cell cycle arrest at G2/M checkpoint associated with a reduced cyclins B1 and B2 expression levels and the upregulation of cell cycle inhibitor p21. Further data suggest the inhibition of Akt and ERK signalling, reduced intercellular cell adhesion molecule-1 and cathepsins expressions, and the presence of cleaved PARP-1, all associated with the observed changes at the cellular level. Gas chromatography-mass spectrometry-based metabolomics reveals distinct metabolite profiles for the juice and vesicle fractions. NVs exhibit a high relative amount of amino acids and organic acids whereas MVs and fruit juice are characterized by a high percentage of sugars and sugar derivatives. Grapefruit-derived NVs are in particular rich in alpha-hydroxy acids and leucine/isoleucine, myo-inositol and doconexent, while quininic acid was detected in MVs. Our findings reveal the metabolite signatures of grapefruit-derived vesicles and substantiate their potential use in new anticancer strategies.

Boosting the Synthesis of Pharmaceutically Active Abietane Diterpenes in S. sclarea Hairy Roots by Engineering the GGPPS and CPPS Genes.

Abietane diterpenoids (ADs), synthesized in the roots of different Salvia species, such as aethiopinone, 1-oxoaethiopinone, salvipisone, and ferruginol, have a variety of known biological activities. We have shown that aethiopinone has promising cytotoxic activity against several human tumor cell lines, including the breast adenocarcinoma MCF7, HeLa, epithelial carcinoma, prostate adenocarcinoma PC3, and human melanoma A375. The low content of these compounds in natural sources, and the limited possibility to synthesize them chemically at low cost, prompted us to optimize the production of abietane diterpenoids by targeting genes of the methylerythritol phosphate (MEP) pathway, from which they are derived. Here, we report our current and ongoing efforts to boost the metabolic flux towards this interesting class of compounds in Salvia sclarea hairy roots (HRs). Silencing the gene encoding the ent-copalyl-diphosphate synthase gene (entCPPS), acting at the lateral geranylgeranyl pyrophosphate (GGPP) competitive gibberellin route, enhanced the content of aethiopinone and other ADs in S. sclarea HRs, indicating indirectly that the GGPP pool is a metabolic constraint to the accumulation of ADs. This was confirmed by overexpressing the GGPPS gene (geranyl-geranyl diphosphate synthase) which triggered also a significant 8-fold increase of abietane diterpene content above the basal constitutive level, with a major boosting effect on aethiopinone accumulation in S. sclarea HRs. A significant accumulation of aethiopinone and other AD compounds was also achieved by overexpressing the CPPS gene (copalyl diphosphate synthase) pointing to this biosynthetic step as another potential metabolic target for optimizing the biosynthesis of this class of compounds. However, by co-expressing of GGPPS and CPPS genes, albeit significant, the increase of abietane diterpenoids was less effective than that obtained by overexpressing the two genes individually. Taken together, the results presented here add novel and instrumental knowledge to a rational design of a hairy root-based platform to yield reliable amounts of aethiopinone and other ADs for a deeper understanding of their molecular pharmacological targets and potential future commercialization.

Nanotechnology in Plant Science: To Make a Long Story Short.

This mini-review aims at gaining knowledge on basic aspects of plant nanotechnology. While in recent years the enormous progress of nanotechnology in biomedical sciences has revolutionized therapeutic and diagnostic approaches, the comprehension of nanoparticle-plant interactions, including uptake, mobilization and accumulation, is still in its infancy. Deeper studies are needed to establish the impact of nanomaterials (NMs) on plant growth and agro-ecosystems and to develop smart nanotechnology applications in crop improvement. Herein we provide a short overview of NMs employed in plant science and concisely describe key NM-plant interactions in terms of uptake, mobilization mechanisms, and biological effects. The major current applications in plants are reviewed also discussing the potential use of polymeric soft NMs which may open new and safer opportunities for smart delivery of biomolecules and for new strategies in plant genetic engineering, with the final aim to enhance plant defense and/or stimulate plant growth and development and, ultimately, crop production. Finally, we envisage that multidisciplinary collaborative approaches will be central to fill the knowledge gap in plant nanotechnology and push toward the use of NMs in agriculture and, more in general, in plant science research.

High Yield of Bioactive Abietane Diterpenes in Salvia sclarea Hairy Roots by Overexpressing Cyanobacterial DXS or DXR Genes.

Abietane diterpenoids, containing a quinone moiety, are synthesized in the roots of several Salvia species. Promising cytotoxicity and antiproliferative activities have been reported for these compounds in various cell and animal models. We have recently shown that aethiopinone, an o-naphto-quinone diterpene, produced in the roots of different Salvia species, is selectively cytotoxic against the A375 melanoma cell line. To enhance the synthesis of this abietane diterpenoid, we have engineered the plastidial 2-C-methyl-D-erythritol 4-phosphate-derived isoprenoid pathway in Salvia sclarea hairy roots by ectopic expression and plastid targeting of cyanobacterial genes encoding the 1-deoxy-D-xylulose 5-phosphate synthase or 1-deoxy-D-xylulose-5-phosphate reductoisomerase gene, the first two enzymatic steps of the plastidial MEP pathway, from which plant diterpenes primarily derive. Plastid-targeted expression of 1-deoxy-D-xylulose 5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase proteins significantly enhanced the yield of aethiopinone by a 3-fold and about 6-fold increase, respectively. The accumulation of other abietane-type diterpenes (ferruginol, salvipisone, and carnosic acid), with interesting antiproliferative activity, was also increased. Compared to our previous data obtained by overexpressing the plant orthologous 1-deoxy-D-xylulose 5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase genes in S. sclarea hairy roots, the results presented here confirm that the bacterial 1-deoxy-D-xylulose-5-phosphate reductoisomerase enzyme plays a major role than the DXS enzyme in the biosynthetic pathway of this class of compounds and that its ectopic expression does not conflict with active hairy root growth, resulting in a balanced trade-off between the transgenic hairy root final biomass and the increased content of o-naphto-quinone diterpenes, with interesting biological activities.

Coactivation of MEP-biosynthetic genes and accumulation of abietane diterpenes in Salvia sclarea by heterologous expression of WRKY and MYC2 transcription factors.

Plant abietane diterpenoids (e.g. aethiopinone, 1- oxoaethiopinone, salvipisone and ferruginol), synthesized in the roots of several Salvia spp, have antibacterial, antifungal, sedative and anti-proliferative properties. Recently we have reported that content of these compounds in S. sclarea hairy roots is strongly depending on transcriptional regulation of genes belonging to the plastidial MEP-dependent terpenoid pathway, from which they mostly derive. To boost the synthesis of this interesting class of compounds, heterologous AtWRKY18, AtWRKY40, and AtMYC2 TFs were overexpressed in S. sclarea hairy roots and proved to regulate in a coordinated manner the expression of several genes encoding enzymes of the MEP-dependent pathway, especially DXS, DXR, GGPPS and CPPS. The content of total abietane diterpenes was enhanced in all overexpressing lines, although in a variable manner due to a negative pleiotropic effect on HR growth. Interestingly, in the best performing HR lines overexpressing the AtWRKY40 TF induced a significant 4-fold increase in the final yield of aethiopinone, for which we have reported an interesting anti-proliferative activity against resistant melanoma cells. The present results are also informative and instrumental to enhance the synthesis of abietane diterpenes derived from the plastidial MEP-derived terpenoid pathway in other Salvia species.

Distinct gene networks drive differential response to abrupt or gradual water deficit in potato.

Water-limiting conditions affect dramatically plant growth and development and, ultimately, yield of potato plants (Solanum tuberosum L.). Therefore, understanding the mechanisms underlying the response to water deficit is of paramount interest to obtain drought tolerant potato varieties. Herein, potato 10K cDNA array slides were used to profile transcriptomic changes of two potato cell populations under abrupt (shocked cells) or gradual exposure (adapted cells) to polyethylene glycol (PEG)-mediated water stress. Data analysis identified >1000 differentially expressed genes (DEGs) in our experimental conditions. Noteworthy, our microarray study also suggests that distinct gene networks underlie the cellular response to shock or gradual water stress. On the basis of our experimental findings, it is possible to speculate that DEGs identified in shocked cells participate in early protective and sensing mechanisms to environmental insults, while the genes whose expression was modulated in adapted cells are directly involved in the acquisition of a new cellular homeostasis to cope with water stress conditions. To validate microarray data obtained for potato cells, the expression analysis of 21 selected genes of interest was performed by Real-Time Quantitative Reverse Transcription PCR (qRT-PCR). Intriguingly, the expression levels of these transcripts in 4-week old potato plants exposed to long-term water-deficit. qRT-PCR analysis showed that several genes were regulated similarly in potato cells cultures and tissues exposed to drought, thus confirming the efficacy of our simple experimental system to capture important genes involved in osmotic stress response. Highlighting the differences in gene expression between shock-like and adaptive response, our findings could contribute to the discussion on the biological function of distinct gene networks involved in the response to abrupt and gradual adaptation to water deficit.

Identification of Limonol Derivatives as Heat Shock Protein†90 (Hsp90) Inhibitors through a Multidisciplinary Approach.

The identification of inhibitors of Hsp90 is currently a primary goal in the development of more effective drugs for the treatment of various types of multidrug resistant malignancies. In an attempt to identify new small molecules modulating the activity of Hsp90, we screened a small library of tetranortriterpenes. A high-affinity interaction with Hsp90 inducible form was uncovered for eight of these compounds, five of which are described here for the first time. By monitoring the ATPase activity and the citrate synthase thermal induced aggregation, compound 1 (cedrelosin A), 3 (7α-limonylacetate), and 5 (cedrelosin B), containing a limonol moiety, were found to be the most effective in compromising the Hsp90α chaperone activity. Consistent with these findings, the three compounds caused a depletion of c-Raf and pAkt Hsp90 client proteins in HeLa and MCF/7 cell lines. Induced fit docking protocol and molecular dynamics were used to rationalize the structural basis of the biological activity of the limonol derivatives. Taken together, these results point to limonol-derivatives as promising scaffolds for the design of novel Hsp90α inhibitors.

The Arabidopsis RNA-binding protein AtRGGA regulates tolerance to salt and drought stress.

Salt and drought stress severely reduce plant growth and crop productivity worldwide. The identification of genes underlying stress response and tolerance is the subject of intense research in plant biology. Through microarray analyses, we previously identified in potato (Solanum tuberosum) StRGGA, coding for an Arginine Glycine Glycine (RGG) box-containing RNA-binding protein, whose expression was specifically induced in potato cell cultures gradually exposed to osmotic stress. Here, we show that the Arabidopsis (Arabidopsis thaliana) ortholog, AtRGGA, is a functional RNA-binding protein required for a proper response to osmotic stress. AtRGGA gene expression was up-regulated in seedlings after long-term exposure to abscisic acid (ABA) and polyethylene glycol, while treatments with NaCl resulted in AtRGGA down-regulation. AtRGGA promoter analysis showed activity in several tissues, including stomata, the organs controlling transpiration. Fusion of AtRGGA with yellow fluorescent protein indicated that AtRGGA is localized in the cytoplasm and the cytoplasmic perinuclear region. In addition, the rgga knockout mutant was hypersensitive to ABA in root growth and survival tests and to salt stress during germination and at the vegetative stage. AtRGGA-overexpressing plants showed higher tolerance to ABA and salt stress on plates and in soil, accumulating lower levels of proline when exposed to drought stress. Finally, a global analysis of gene expression revealed extensive alterations in the transcriptome under salt stress, including several genes such as ASCORBATE PEROXIDASE2, GLUTATHIONE S-TRANSFERASE TAU9, and several SMALL AUXIN UPREGULATED RNA-like genes showing opposite expression behavior in transgenic and knockout plants. Taken together, our results reveal an important role of AtRGGA in the mechanisms of plant response and adaptation to stress.

Targeting the Hsp90 C-terminal domain by the chemically accessible dihydropyrimidinone scaffold.

Hsp90 C-terminal ligands are potential new anti-cancer drugs alternative to the more studied N-terminal inhibitors. Here we report the identification of a new dihydropyrimidinone binding the C-terminus, which is not structurally related to other well-known natural and nature-inspired inhibitors of this second druggable Hsp90 site.