Farming for Pharming: Novel Hydroponic Process in Contained Environment for Efficient Pharma-Grade Production of Saffron.

Soilless cultivation of saffron (Crocus sativus) in a controlled environment represents an interesting alternative to field cultivation, in order to obtain a standardized high-quality product and to optimize yields. In particular, pharma-grade saffron is fundamental for therapeutic applications of this spice, whose efficacy has been demonstrated in the treatment of macular diseases, such as Age-related Macular Degeneration (AMD). In this work, a hydroponic cultivation system was developed, specifically designed to meet the needs of C. sativus plant. Various cultivation recipes, different in spectrum and intensity of lighting, temperature, photoperiod and irrigation, have been adopted to study their effect on saffron production. The experimentation involved the cultivation of corms from two subsequent farm years, to identify and validate the optimal conditions, both in terms of quantitative yield and as accumulation of bioactive metabolites, with particular reference to crocins and picrocrocin, which define the 'pharma-grade' quality of saffron. Through HPLC analysis and chromatography it was possible to identify the cultivation parameters suitable for the production of saffron with neuroprotective properties, evaluated by comparison with an ISO standard and the REPRON® procedure. Furthermore, the biochemical characterization was completed through NMR and high-resolution mass spectrometry analyses of saffron extracts. The whole experimental framework allowed to establish an optimized protocol to produce pharma-grade saffron, allowing up to 3.2 g/m2 harvest (i.e., more than three times higher than field production in optimal conditions), which meets the standards of composition for the therapy of AMD.

Beyond DNA repair, the immunological role of PARP-1 and its siblings.

ADP-ribosylation is the addition of one or more (up to some hundreds) ADP-ribose moieties to acceptor proteins. There are two major families of enzymes that catalyse this reaction: extracellular ADP-ribosyl-transferases (ARTs), which are bound to the cell membrane by a glycosylphosphatidylinositol anchor or are secreted, and poly(ADP-ribose)-polymerases (PARPs), which are present in the cell nucleus and/or cytoplasm. Recent findings revealed a wide immunological role for ADP-ribosylating enzymes. ARTs, by sensing extracellular NAD concentration, can act as danger detectors. PARP-1, the prototypical representative of the PARP family, known to protect cells from genomic instability, is involved in the development of inflammatory responses and several forms of cell death. PARP-1 also plays a role in adaptive immunity by modulating the ability of dendritic cells to stimulate T cells or by directly affecting the differentiation and functions of T and B cells. Both PARP-1 and PARP-14 (CoaSt6) knockout mice were described to display reduced T helper type 2 cell differentiation and allergic responses. Our recent findings showed that PARP-1 is involved in the differentiation of Foxp3+ regulatory T (Treg) cells, suggesting a role for PARP-1 in tolerance induction. Also ARTs regulate Treg cell homeostasis by promoting Treg cell apoptosis during inflammatory responses. PARP inhibitors ameliorate immune-mediated diseases in several experimental models, including rheumatoid arthritis, colitis, experimental autoimmune encephalomyelitis and allergy. Together these findings show that ADP-ribosylating enzymes, in particular PARP-1, play a pivotal role in the regulation of immune responses and may represent a good target for new therapeutic approaches in immune-mediated diseases.

Effects of gamma radiation on engineered tomato biofortified for space agriculture by morphometry and fluorescence-based indices.

Introduction: Future long-term space missions will focus to the solar system exploration, with the Moon and Mars as leading goals. Plant cultivation will provide fresh food as a healthy supplement to astronauts' diet in confined and unhealthy outposts. Ionizing radiation (IR) are a main hazard in outer space for their capacity to generate oxidative stress and DNA damage. IR is a crucial issue not only for human survival, but also for plant development and related value-added fresh food harvest. To this end, efforts to figure out how biofortification of plants with antioxidant metabolites (such as anthocyanins) may contribute to improve their performances in space outposts are needed. Methods: MicroTom plants genetically engineered to express the Petunia hybrida PhAN4 gene, restoring the biosynthesis of anthocyanins in tomato, were used. Seeds and plants from wild type and engineered lines AN4-M and AN4-P2 were exposed to IR doses that they may experience during a long-term space mission, simulated through the administration of gamma radiation. Plant response was continuously evaluated along life cycle by a non-disturbing/non-destructive monitoring of biometric and multiparametric fluorescence-based indices at both phenotypic and phenological levels, and indirectly measuring changes occurring at the primary and secondary metabolism level. Results: Responses to gamma radiation were influenced by the phenological stage, dose and genotype. Wild type and engineered plants did not complete a seed-to-seed cycle under the exceptional condition of 30 Gy absorbed dose, but were able to cope with 0.5 and 5 Gy producing fruits and vital seeds. In particular, the AN4-M seeds and plants showed advantages over wild type: negligible variation of fluorimetric parameters related to primary metabolism, no alteration or improvement of yield traits at maturity while maintaining smaller habitus than wild type, biosynthesis of anthocyanins and maintained levels of these compounds compared to non-irradiated controls of the same age. Discussion: These findings may be useful in understanding phenotypic effects of IR on plant growth in space, and lead to the exploitation of new breeding efforts to optimize plant performances to develop appropriate ideotypes for future long-term space exploration extending the potential of plants to serve as high-value product source.

Non-destructive real-time analysis of plant metabolite accumulation in radish microgreens under different LED light recipes.

Introduction: The future of human space missions relies on the ability to provide adequate food resources for astronauts and also to reduce stress due to the environment (microgravity and cosmic radiation). In this context, microgreens have been proposed for the astronaut diet because of their fast-growing time and their high levels of bioactive compounds and nutrients (vitamins, antioxidants, minerals, etc.), which are even higher than mature plants, and are usually consumed as ready-to-eat vegetables. Methods: Our study aimed to identify the best light recipe for the soilless cultivation of two cultivars of radish microgreens (Raphanus sativus, green daikon, and rioja improved) harvested eight days after sowing that could be used for space farming. The effects on plant metabolism of three different light emitting diodes (LED) light recipes (L1-20% red, 20% green, 60% blue; L2-40% red, 20% green, 40% blue; L3-60% red, 20% green, 20% blue) were tested on radish microgreens hydroponically grown. A fluorimetric-based technique was used for a real-time non-destructive screening to characterize plant methabolism. The adopted sensors allowed us to quantitatively estimate the fluorescence of flavonols, anthocyanins, and chlorophyll via specific indices verified by standardized spectrophotometric methods. To assess plant growth, morphometric parameters (fresh and dry weight, cotyledon area and weight, hypocotyl length) were analyzed. Results: We observed a statistically significant positive effect on biomass accumulation and productivity for both cultivars grown under the same light recipe (40% blue, 20% green, 40% red). We further investigated how the addition of UV and/or far-red LED lights could have a positive effect on plant metabolite accumulation (anthocyanins and flavonols). Discussion: These results can help design plant-based bioregenerative life-support systems for long-duration human space exploration, by integrating fluorescence-based non-destructive techniques to monitor the accumulation of metabolites with nutraceutical properties in soilless cultivated microgreens.

Modifying Anthocyanins Biosynthesis in Tomato Hairy Roots: A Test Bed for Plant Resistance to Ionizing Radiation and Antioxidant Properties in Space.

Gene expression manipulation of specific metabolic pathways can be used to obtain bioaccumulation of valuable molecules and desired quality traits in plants. A single-gene approach to impact different traits would be greatly desirable in agrospace applications, where several aspects of plant physiology can be affected, influencing growth. In this work, MicroTom hairy root cultures expressing a MYB-like transcription factor that regulates the biosynthesis of anthocyanins in Petunia hybrida (PhAN4), were considered as a testbed for bio-fortified tomato whole plants aimed at agrospace applications. Ectopic expression of PhAN4 promoted biosynthesis of anthocyanins, allowing to profile 5 major derivatives of delphinidin and petunidin together with pelargonidin and malvidin-based anthocyanins, unusual in tomato. Consistent with PhAN4 features, transcriptomic profiling indicated upregulation of genes correlated to anthocyanin biosynthesis. Interestingly, a transcriptome reprogramming oriented to positive regulation of cell response to biotic, abiotic, and redox stimuli was evidenced. PhAN4 hairy root cultures showed the significant capability to counteract reactive oxygen species (ROS) accumulation and protein misfolding upon high-dose gamma irradiation, which is among the most potent pro-oxidant stress that can be encountered in space. These results may have significance in the engineering of whole tomato plants that can benefit space agriculture.

CultCube: Experiments in autonomous in-orbit cultivation on-board a 12-Units CubeSat platform.

The feasibility and design of the CultCube 12U CubeSat hosting a small Environmental Control and Life Support Systems (ECLSS) for the autonomous cultivation of a small plant in orbit is described. The satellite is aimed at running experiments in fruit plants growing for applications in crewed vehicles for long-term missions in space. CultCube is mainly composed of a pressurized vessel, constituting the outer shell of the ECLSS, and by various environmental controls (water, nutrients, air composition and pressure, light, etc.) aimed at maintaining a survivable habitat for the fruit plants to grow. The plant health status and growth performances is monitored using hyperspectral cameras installed within the vessel, able to sense leaves' chlorophyll content and temperature, and allowing the estimation of plant volume in all its life cycle phases. The paper study case is addressed to the in-orbit experimental cultivation of a dwarf tomato plant (MicroTom), which was modified for enhancing the anti-oxidants production and for growing in stressful environments. While simulated microgravity tests have been passed by the MicroTom plant, the organism behaviour in a real microgravity environment for a full seed-to-seed cycle needs to be tested. The CultCube 12U CubeSat mission presents no particular requirements on the kind of orbit, whereas its minimum significative duration corresponds to one seed-to-seed cycle for the plant, which is 90 days for the paper study case. In the paper, after an introduction on the importance of an autonomous testbed for plant cultivation, in the perspective of the implementation of bioregenerative systems on-board future manned long-term missions, the satellite design and the MicroTom engineered plant for in-orbit growth are described. In addition to the description of the whole set of subsystems, with focus on the payload and its controllers and instrumentation, the system budgets are presented. Finally, the first tests conducted by the authors are briefly reported.