ß-Hexachlorocyclohexane triggers neuroinflammatory activity, epigenetic histone post-translational modifications and cognitive dysfunction.

Persistent organic pollutants (POPs), which encompass pesticides and industrial chemicals widely utilized across the globe, pose a covert threat to human health. ß-hexachlorocyclohexane (ß-HCH) is an organochlorine pesticide with striking stability, still illegally dumped in many countries, and recognized as responsible for several pathogenetic mechanisms. This study represents a pioneering exploration into the neurotoxic effects induced by the exposure to ß-HCH specifically targeting neuronal cells (N2a), microglia (BV-2), and C57BL/6 mice. As shown by western blot and qPCR analyses, the administration of ß-HCH triggered a modulation of NF-κB, a key factor influencing both inflammation and pro-inflammatory cytokines expression. We demonstrated by proteomic and western blot techniques epigenetic modifications in H3 histone induced by ß-HCH. Histone acetylation of H3K9 and H3K27 increased in N2a, and in the prefrontal cortex of C57BL/6 mice administered with ß-HCH, whereas it decreased in BV-2 cells and in the hippocampus. We also observed a severe detrimental effect on recognition memory and spatial navigation by the Novel Object Recognition Test (NORT) and the Object Place Recognition Task (OPRT) behavioural tests. Cognitive impairment was linked to decreased expression of the genes BDNF and SNAP-25, which are mediators involved in synaptic function and activity. The obtained results expand our understanding of the harmful impact produced by ß-HCH exposure by highlighting its implication in the pathogenesis of neurological diseases. These findings will support intervention programs to limit the risk induced by exposure to POPs. Regulatory agencies should block further illicit use, causing environmental hazards and endangering human and animal health.

Anti-Biofilm Activity of Oleacein and Oleocanthal from Extra-Virgin Olive Oil toward Pseudomonas aeruginosa.

New antimicrobial molecules effective against Pseudomonas aeruginosa, known as an antibiotic-resistant "high-priority pathogen", are urgently required because of its ability to develop biofilms related to healthcare-acquired infections. In this study, for the first time, the anti-biofilm and anti-virulence activities of a polyphenolic extract of extra-virgin olive oil as well as purified oleocanthal and oleacein, toward P. aeruginosa clinical isolates were investigated. The main result of our study was the anti-virulence activity of the mixture of oleacein and oleocanthal toward multidrug-resistant and intermediately resistant strains of P. aeruginosa isolated from patients with ventilator-associated pneumonia or surgical site infection. Specifically, the mixture of oleacein (2.5 mM)/oleocanthal (2.5 mM) significantly inhibited biofilm formation, alginate and pyocyanin production, and motility in both P. aeruginosa strains (p < 0.05); scanning electron microscopy analysis further evidenced its ability to inhibit bacterial cell adhesion as well as the production of the extracellular matrix. In conclusion, our results suggest the potential application of the oleacein/oleocanthal mixture in the management of healthcare-associated P. aeruginosa infections, particularly in the era of increasing antimicrobial resistance.

Natural deep eutectic solvents protect RNA from thermal-induced degradation.

RNA is a fundamental nucleic acid for life and it plays important roles in the regulation of gene transcription, post-transcriptional regulation, and epigenetic regulation. Recently, the focus on this nucleic acid has significantly increased due to the development of mRNA vaccines and RNA-based gene therapy protocols. Unfortunately, RNA based products show constrains mainly owing to instability and easy degradability of the RNA molecules. Indeed, unlike the DNA molecule which has a great intrinsic stability, RNA is more prone to degradation and this process is accelerated under thermal treatment. Here we describe a method that involves the use of Natural Deep Eutectic Solvents (NaDES) capable of slowing down RNA degradation process. Our results show that this technology seems suitable for improving the stability of specific RNA molecules particularly susceptible to thermal-induced degradation. Therefore, this technique represents a valuable tool to stabilize RNA molecules used in gene therapy and mRNA vaccines.

Extra Virgin Olive Oil-Based Formulations: A "Green" Strategy against Chlamydia trachomatis.

In recent decades, antibiotic misuse has emerged as an important risk factor for the appearance of multi-drug-resistant bacteria, and, recently, antimicrobial resistance has also been described in Chlamydia trachomatis as the leading cause of bacterial sexually transmitted diseases worldwide. Herein, we investigated, for the first time, the antibacterial activity against C. trachomatis of a polyphenolic extract of extra virgin olive oil (EVOO), alongside purified oleocanthal and oleacein, two of its main components, in natural deep eutectic solvent (NaDES), a biocompatible solvent. The anti-chlamydial activity of olive-oil polyphenols (OOPs) was tested in the different phases of chlamydial developmental cycle by using an in vitro infection model. Transmission and scanning electron microscopy analysis were performed for investigating potential alterations of adhesion and invasion, as well as morphology, of chlamydial elementary bodies (EBs) to host cells. The main result of our study is the anti-bacterial activity of OOPs towards C. trachomatis EBs down to a total polyphenol concentration of 1.7 µg/mL, as shown by a statistically significant decrease (93.53%) of the total number of chlamydial-inclusion-forming units (p < 0.0001). Transmission and scanning electron microscopy analysis supported its anti-chlamydial effect, suggesting that OOP might damage the chlamydial outer layers, impairing their structural integrity and hindering EB capability to infect the host cell. In conclusion, OOPs may represent an interesting alternative therapeutic option toward C. trachomatis, although further studies are necessary for exploring its clinical applications.

ERp57 chaperon protein protects neuronal cells from Aß-induced toxicity.

Alzheimer's disease (AD) is a neurodegenerative disorder whose main pathological hallmark is the accumulation of Amyloid-ß peptide (Aß) in the form of senile plaques. Aß can cause neurodegeneration and disrupt cognitive functions by several mechanisms, including oxidative stress. ERp57 is a protein disulfide isomerase involved in the cellular stress response and known to be present in the cerebrospinal fluid of normal individuals as a complex with Aß peptides, suggesting that it may be a carrier protein which prevents aggregation of Aß. Although several studies show ERp57 involvement in neurodegenerative diseases, no clear mechanism of action has been identified thus far. In this work, we gain insights into the interaction of Aß with ERp57, with a special focus on the contribution of ERp57 to the defense system of the cell. Here, we show that recombinant ERp57 directly interacts with the Aß25-35 fragment in vitro with high affinity via two in silico-predicted main sites of interaction. Furthermore, we used human neuroblastoma cells to show that short-term Aß25-35 treatment induces ERp57 decrease in intracellular protein levels, different intracellular localization, and ERp57 secretion in the cultured medium. Finally, we demonstrate that recombinant ERp57 counteracts the toxic effects of Aß25-35 and restores cellular viability, by preventing Aß25-35 aggregation. Overall, the present study shows that extracellular ERp57 can exert a protective effect from Aß toxicity and highlights it as a possible therapeutic tool in the treatment of AD.

Taxanes in cancer treatment: Activity, chemoresistance and its overcoming.

Since 1984, when paclitaxel was approved by the FDA for the treatment of advanced ovarian carcinoma, taxanes have been widely used as microtubule-targeting antitumor agents. However, their historic classification as antimitotics does not describe all their functions. Indeed, taxanes act in a complex manner, altering multiple cellular oncogenic processes including mitosis, angiogenesis, apoptosis, inflammatory response, and ROS production. On the one hand, identification of the diverse effects of taxanes on oncogenic signaling pathways provides opportunities to apply these cytotoxic drugs in a more rational manner. On the other hand, this may facilitate the development of novel treatment modalities to surmount anticancer drug resistance. In the latter respect, chemoresistance remains a major impediment which limits the efficacy of antitumor chemotherapy. Taxanes have shown impact on key molecular mechanisms including disruption of mitotic spindle, mitosis slippage and inhibition of angiogenesis. Furthermore, there is an emerging contribution of cellular processes including autophagy, oxidative stress, epigenetic alterations and microRNAs deregulation to the acquisition of taxane resistance. Hence, these two lines of findings are currently promoting a more rational and efficacious taxane application as well as development of novel molecular strategies to enhance the efficacy of taxane-based cancer treatment while overcoming drug resistance. This review provides a general and comprehensive picture on the use of taxanes in cancer treatment. In particular, we describe the history of application of taxanes in anticancer therapeutics, the synthesis of the different drugs belonging to this class of cytotoxic compounds, their features and the differences between them. We further dissect the molecular mechanisms of action of taxanes and the molecular basis underlying the onset of taxane resistance. We further delineate the possible modalities to overcome chemoresistance to taxanes, such as increasing drug solubility, delivery and pharmacokinetics, overcoming microtubule alterations or mitotic slippage, inhibiting drug efflux pumps or drug metabolism, targeting redox metabolism, immune response, and other cellular functions.

Mechano-Transduction Boosts the Aging Effects in Human Erythrocytes Submitted to Mechanical Stimulation.

Erythrocytes' aging and mechano-transduction are fundamental cellular pathways that determine the red blood cells' (RBCs) behavior and function. The aging pattern can be influenced, in morphological, biochemical, and metabolic terms by the environmental conditions. In this paper, we studied the effect of a moderate mechanical stimulation applied through external shaking during the RBCs aging and revealed a strong acceleration of the aging pattern induced by such stimulation. Moreover, we evaluated the behavior of the main cellular effectors and resources in the presence of drugs (diamide) or of specific inhibitors of the mechano-transduction (probenecid, carbenoxolone, and glibenclamide). This approach provided the first evidence of a direct cross-correlation between aging and mechano-transduction and permitted an evaluation of the overall metabolic regulation and of the insurgence of specific morphological features, such as micro-vesicles and roughness alterations. Overall, for the first time the present data provided a schematic to understand the integration of distinct complex patterns in a comprehensive view of the cell and of its interactions with the environment. Mechano-transduction produces structural effects that are correlated with the stimulation and the strength of the environmental stimulation is paramount to effectively activate and trigger the biological cascades initiated by the mechano-sensing.

Effect of Natural Deep Eutectic Solvents on trans-Resveratrol Photo-Chemical Induced Isomerization and 2,4,6-Trihydroxyphenanthrene Electro-Cyclic Formation.

trans-Resveratrol is a natural bioactive compound with well-recognized health promoting effects. When exposed to UV light, this compound can undergo a photochemically induced trans/cis isomerization and a 6π electrochemical cyclization with the subsequent formation of 2,4,6-trihydroxyphenanthrene (THP). THP is a potentially harmful compound which can exert genotoxic effects. In this work we improved the chromatographic separation and determination of the two resveratrol isomers and of THP by using a non-commercial pentafluorophenyl stationary phase. We assessed the effect of natural deep eutectic solvents (NaDES) as possible photo-protective agents by evaluating cis-resveratrol isomer and THP formation under different UV-light exposure conditions with the aim of enhancing resveratrol photostability and inhibiting THP production. Our results demonstrate a marked photoprotective effect exerted by glycerol-containing NaDES, and in particular by proline/glycerol NaDES, which exerts a strong inhibitory effect on the photochemical isomerization of resveratrol and significantly limits the formation of the toxic derivative THP. Considering the presence of resveratrol in various commercial products, these results are of note in view of the potential genotoxic risk associated with its photochemical degradation products and in view of the need for the development of green, eco-sustainable and biocompatible resveratrol photo-stable formulations.

Pharmacokinetic properties of a novel formulation of S-adenosyl-L-methionine phytate.

S-adenosyl-L-methionine (SAM), the main endogenous methyl donor, is the adenosyl derivative of the amino acid methionine, which displays many important roles in cellular metabolism. It is widely used as a food supplement and in some countries is also marketed as a drug. Its interesting nutraceutical and pharmacological properties prompted us to evaluate the pharmacokinetics of a new form of SAM, the phytate salt. The product was administered orally to rats and pharmacokinetic parameters were evaluated by comparing the results with that obtained by administering the SAM tosylated form (SAM PTS). It was found that phytate anion protects SAM from degradation, probably because of steric hindrance exerted by the counterion, and that the SAM phytate displayed significant better pharmacokinetic parameters compared to SAM PTS. These results open to the perspective of the use of new salts of SAM endowed with better pharmacokinetic properties.

Pheomelanin Effect on UVB Radiation-Induced Oxidation/Nitration of l-Tyrosine.

Pheomelanin is a natural yellow-reddish sulfur-containing pigment derived from tyrosinase-catalyzed oxidation of tyrosine in presence of cysteine. Generally, the formation of melanin pigments is a protective response against the damaging effects of UV radiation in skin. However, pheomelanin, like other photosensitizing substances, can trigger, following exposure to UV radiation, photochemical reactions capable of modifying and damaging cellular components. The photoproperties of this natural pigment have been studied by analyzing pheomelanin effect on oxidation/nitration of tyrosine induced by UVB radiation at different pH values and in presence of iron ions. Photoproperties of pheomelanin can be modulated by various experimental conditions, ranging from the photoprotection to the triggering of potentially damaging photochemical reactions. The study of the photomodification of l-Tyrosine in the presence of the natural pigment pheomelanin has a special relevance, since this tyrosine oxidation/nitration pathway can potentially occur in vivo in tissues exposed to sunlight and play a role in the mechanisms of tissue damage induced by UV radiation.

Biochemical and Computational Studies of the Interaction between a Glucosamine Derivative, NAPA, and the IKKα Kinase.

The glucosamine derivative 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-ß-D-glucose (NAPA), was shown to inhibit the kinase activity of IKKα, one of the two catalytic subunits of IKK complex, decreasing the inflammatory status in osteoarthritis chondrocytes. In the present work we have investigated the inhibition mechanism of IKKα by NAPA by combining computational simulations, in vitro assays and Mass Spectrometry (MS) technique. The kinase in vitro assay was conducted using a recombinant IKKα and IKKtide, a 20 amino acid peptide substrate derived from IkBα kinase protein and containing the serine residues Ser32 and Ser36. Phosphorylated peptide production was measured by Ultra Performance Liquid Chromatography coupled with Mass Spectrometry (UPLC-MS), and the atomic interaction between IKKα and NAPA has been studied by molecular docking and Molecular Dynamics (MD) approaches. Here we report that NAPA was able to inhibit the IKKα kinase activity with an IC50 of 0.5 mM, to decrease the Km value from 0.337 mM to 0.402 mM and the Vmax from 0.0257 mM·min-1 to 0.0076 mM·min-1. The computational analyses indicate the region between the KD, ULD and SDD domains of IKKα as the optimal binding site explored by NAPA. Biochemical data indicate that there is a non-significant difference between Km and Ki whereas there is a statistically significant difference between the two Vmax values. This evidence, combined with computational results, consistently indicates that the inhibition is non-competitive, and that the NAPA binding site is different than that of ATP or IKKtide.

Editorial for Special Issue: Anthocyanin.

What do jabuticaba (Myrciaria jaboticaba), Jamun berry (Syzygium cumini), Malay apple (Syzygium malaccense), crimson glory vine (Vitis coignetiae) and roselle (Hibiscus sabdariffa) have in common [...].

GO Nanosheets: Promising Nano Carrier for the S29, 1-(2-Chloro-2-(4-chlorophenyl-ethyl)-N-(4-fluorobenzyl)-1H-pyrazolo[3,4-d] pyrimidin-4-amine, Therapeutic Agent in Neuroblastoma.

Graphene oxide (GO) derivatives are reported as a valid alternative to conventional carriers of therapeutic agents, because they have a large surface area, an excellent electrical and thermal conductivity and a great capacity for selective binding of drugs and therapeutics, due to the functionalization of their surfaces, edges and sides. In this work GO nanosheets, synthesized by electrochemical exfoliation of graphite (patent N 102015000023739, Tor Vergata University), were investigated as possible carriers of an anticancer drug, the S29, an inhibitor of a cytoplasmic tyrosine kinase (c-SRC) on a neuroblastoma cell line (SK N BE 2 cells). Neuroblastoma is a heterogenous tumor whose characteristics range from spontaneous regression to aggressive phenotypes that are due to different mutations that often occur in SRC family kinases. Inhibitors of tyrosine kinases are currently investigated for their anti-tumoral effects on aggressive neuroblastomas, but their uptake in cells and pharmacokinetics needs to be improved. In this work S29 was stably conjugated with highly water-dispersible GO nanoparticles. S29/GO complex formation was induced by 1h sonication and its stability was analyzed by chromatography coupled with spectrophotometry and mass spectrometry. The synthesized composite (GO-S29) was delivered into SK N BE 2 cells and its effects on cell viability, production of reactive oxygen species (ROS) and migration were studied. The results show that the compound GO-S29 exerts anti-tumoral effects on the neuroblastoma cell line, higher than both GO and S29 do alone and that GO has an additive effect on S29.

Anthocyanins: A Comprehensive Review of Their Chemical Properties and Health Effects on Cardiovascular and Neurodegenerative Diseases.

Anthocyanins are a class of water-soluble flavonoids widely present in fruits and vegetables. Dietary sources of anthocyanins include red and purple berries, grapes, apples, plums, cabbage, or foods containing high levels of natural colorants. Cyanidin, delphinidin, malvidin, peonidin, petunidin, and pelargonidin are the six common anthocyanidins. Following consumption, anthocyanin, absorption occurs along the gastrointestinal tract, the distal lower bowel being the place where most of the absorption and metabolism occurs. In the intestine, anthocyanins first undergo extensive microbial catabolism followed by absorption and human phase II metabolism. This produces hybrid microbial-human metabolites which are absorbed and subsequently increase the bioavailability of anthocyanins. Health benefits of anthocyanins have been widely described, especially in the prevention of diseases associated with oxidative stress, such as cardiovascular and neurodegenerative diseases. Furthermore, recent evidence suggests that health-promoting effects attributed to anthocyanins may also be related to modulation of gut microbiota. In this paper we attempt to provide a comprehensive view of the state-of-the-art literature on anthocyanins, summarizing recent findings on their chemistry, biosynthesis, nutritional value and on their effects on human health.

Green Route for the Isolation and Purification of Hyrdoxytyrosol, Tyrosol, Oleacein and Oleocanthal from Extra Virgin Olive Oil.

Extra virgin olive oil (EVOO) phenols represent a significant part of the intake of antioxidants and bioactive compounds in the Mediterranean diet. In particular, hydroxytyrosol (HTyr), tyrosol (Tyr), and the secoiridoids oleacein and oleocanthal play central roles as anti-inflammatory, neuro-protective and anti-cancer agents. These compounds cannot be easily obtained via chemical synthesis, and their isolation and purification from EVOO is cumbersome. Indeed, both processes involve the use of large volumes of organic solvents, hazardous reagents and several chromatographic steps. In this work we propose a novel optimized procedure for the green extraction, isolation and purification of HTyr, Tyr, oleacein and oleocanthal directly from EVOO, by using a Natural Deep Eutectic Solvent (NaDES) as an extracting phase, coupled with preparative high-performance liquid chromatography. This purification method allows the total recovery of the four components as single pure compounds directly from EVOO, in a rapid, economic and ecologically sustainable way, which utilizes biocompatible reagents and strongly limits the use or generation of hazardous substances.

One- and Two-Electron Oxidations of ß-Amyloid25-35 by Carbonate Radical Anion (CO3•-) and Peroxymonocarbonate (HCO4-): Role of Sulfur in Radical Reactions and Peptide Aggregation.

The ß-amyloid (Aß) peptide plays a key role in the pathogenesis of Alzheimer's disease. The methionine (Met) residue at position 35 in Aß C-terminal domain is critical for neurotoxicity, aggregation, and free radical formation initiated by the peptide. The role of Met in modulating toxicological properties of Aß most likely involves an oxidative event at the sulfur atom. We therefore investigated the one- or two-electron oxidation of the Met residue of Aß25-35 fragment and the effect of such oxidation on the behavior of the peptide. Bicarbonate promotes two-electron oxidations mediated by hydrogen peroxide after generation of peroxymonocarbonate (HCO4-, PMC). The bicarbonate/carbon dioxide pair stimulates one-electron oxidations mediated by carbonate radical anion (CO3•-). PMC efficiently oxidizes thioether sulfur of the Met residue to sulfoxide. Interestingly, such oxidation hampers the tendency of Aß to aggregate. Conversely, CO3•- causes the one-electron oxidation of methionine residue to sulfur radical cation (MetS•+). The formation of this transient reactive intermediate during Aß oxidation may play an important role in the process underlying amyloid neurotoxicity and free radical generation.

Thiotaurine: From Chemical and Biological Properties to Role in H2S Signaling.

In the last decade thiotaurine, 2-aminoethane thiosulfonate, has been investigated as an inflammatory modulating agent as a result of its ability to release hydrogen sulfide (H2S) known to play regulatory roles in inflammation. Thiotaurine can be included in the "taurine family" due to structural similarity to taurine and hypotaurine, and is characterized by the presence of a sulfane sulfur moiety. Thiotaurine can be produced by different pathways, such as the spontaneous transsulfuration between thiocysteine - a persulfide analogue of cysteine - and hypotaurine as well as in vivo from cystine. Moreover, the enzymatic oxidation of cysteamine to hypotaurine and thiotaurine in the presence of inorganic sulfur can occur in animal tissues and last but not least thiotaurine can be generated by the transfer of sulfur from mercaptopyruvate to hypotaurine catalyzed by a sulfurtransferase. Thiotaurine is an effective antioxidant agent as demonstrated by its ability to counteract the damage caused by pro-oxidants in the rat. Recently, we observed the influence of thiotaurine on human neutrophils functional responses. In particular, thiotaurine has been found to prevent human neutrophil spontaneous apoptosis suggesting an alternative or additional role to its antioxidant activity. It is likely that the sulfane sulfur of thiotaurine may modulate neutrophil activation via persulfidation of target proteins. In conclusion, thiotaurine can represent a biologically relevant sulfur donor acting as a biological intermediate in the transport, storage and release of sulfide.

Anti-Inflammatory Activity of A Polyphenolic Extract from Arabidopsis thaliana in In Vitro and In Vivo Models of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common neurodegenerative disorder and the primary form of dementia in the elderly. One of the main features of AD is the increase in amyloid-beta (Aß) peptide production and aggregation, leading to oxidative stress, neuroinflammation and neurodegeneration. Polyphenols are well known for their antioxidant, anti-inflammatory and neuroprotective effects and have been proposed as possible therapeutic agents against AD. Here, we investigated the effects of a polyphenolic extract of Arabidopsis thaliana (a plant belonging to the Brassicaceae family) on inflammatory response induced by Aß. BV2 murine microglia cells treated with both Aß25⁻35 peptide and extract showed a lower pro-inflammatory (IL-6, IL-1ß, TNF-α) and a higher anti-inflammatory (IL-4, IL-10, IL-13) cytokine production compared to cells treated with Aß only. The activation of the Nrf2-antioxidant response element signaling pathway in treated cells resulted in the upregulation of heme oxygenase-1 mRNA and in an increase of NAD(P)H:quinone oxidoreductase 1 activity. To establish whether the extract is also effective against Aß-induced neurotoxicity in vivo, we evaluated its effect on the impaired climbing ability of AD Drosophila flies expressing human Aß1⁻42. Arabidopsis extract significantly restored the locomotor activity of these flies, thus confirming its neuroprotective effects also in vivo. These results point to a protective effect of the Arabidopsis extract in AD, and prompt its use as a model in studying the impact of complex mixtures derived from plant-based food on neurodegenerative diseases.

Insights into the Phytochemistry of the Cuban Endemic Medicinal Plant Phyllanthus orbicularis: Fideloside, a Novel Bioactive 8-C-glycosyl 2,3-Dihydroflavonol.

Phyllanthus orbicularis (Phyllanthaceae) is an endemic evergreen tropical plant of Cuba that grows in the western part of the island and is used in traditional medicine as an infusion. The aqueous extract of this plant presents a wide range of pharmacological activitiessuch as antimutagenic, antioxidant and antiviral effects. Given the many beneficial effects and the great interest in the development of new pharmacological products from natural sources, the aim of this work was to investigate the phytochemistry of this species and to elucidate the structure of the main bioactive principles. Besides the presence of several known polyphenols, the major constituent was hitherto not described. The chemical structure of this compound, here named Fideloside, was elucidated by means of HR-ESIMS/MSn, 1D/2D NMR, FT-IR, and ECD as (2R,3R)-(-)-3',4',5,7-tetrahydroxydihydroflavonol-8-C-ß-D-glucopyranoside. The compound, as well as the plant aqueous preparations, showed promising bioactive properties, i.e., anti-inflammatory capacity in human explanted monocytes, corroborating future pharmacological use for this new natural C-glycosyl flavanonol.

Proline synthesis in developing microspores is required for pollen development and fertility.

BACKGROUND: In many plants, the amino acid proline is strongly accumulated in pollen and disruption of proline synthesis caused abortion of microspore development in Arabidopsis. So far, it was unclear whether local biosynthesis or transport of proline determines the success of fertile pollen development. RESULTS: We analyzed the expression pattern of the proline biosynthetic genes PYRROLINE-5-CARBOXYLATE SYNTHETASE 1 & 2 (P5CS1 & 2) in Arabidopsis anthers and both isoforms were strongly expressed in developing microspores and pollen grains but only inconsistently in surrounding sporophytic tissues. We introduced in a p5cs1/p5cs1 p5cs2/P5CS2 mutant background an additional copy of P5CS2 under the control of the Cauliflower Mosaic Virus (CaMV) 35S promoter, the tapetum-specific LIPID TRANSFER PROTEIN 12 (Ltp12) promoter or the pollen-specific At5g17340 promoter to determine in which site proline biosynthesis can restore the fertility of proline-deficient microspores. The specificity of these promoters was confirmed by ß-glucuronidase (GUS) analysis, and by direct proline measurement in pollen grains and stage-9/10 anthers. Expression of P5CS2 under control of the At5g17340 promoter fully rescued proline content and normal morphology and fertility of mutant pollen. In contrast, expression of P5CS2 driven by either the Ltp12 or CaMV35S promoter caused only partial restoration of pollen development with little effect on pollen fertility. CONCLUSIONS: Overall, our results indicate that proline transport is not able to fulfill the demand of the cells of the male germ line. Pollen development and fertility depend on local proline biosynthesis during late stages of microspore development and in mature pollen grains.