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Plants have always been a source of metabolic compounds useful for human health. Today, the different genetic engineering techniques allow us to modify both whole plants and in vitro cultured tissues and organs for several purposes. These span from the elicitation of bioactive molecules that are already produced by these organisms to de novo biosynthesis of metabolites and recombinant proteins (Burnett et al., 2019). In particular, several studies have focused on the production of compounds with antioxidant, anti-inflammatory and hypoglycemic activity with the long term goals of exploiting their protective role in the prevention of neurodegenerative diseases and cancer, just to cite a few (Zhang et al., 2015). Indeed, plant in vitro systems appear to be reliable and sustainable platforms for the production of such as molecules (Marchev and Georgiev, 2020). Here we show three strategies we are currently pursuing for the production of compounds with antioxidant or immunomodulatory actions in planta. They are based either on stable or transient genetic transformation of plant cells from (a) Malus domestica, (b) Daucus carota and (c) Nicotiana tabacum. In (a) calli derived from apple pulp have been transformedwith the bHLH transcription factor Sn (TF) from Zea mays. This TF interacts with MYB and WD40 proteinsto upregulate the biosynthesis of anthocyanins and/or proanthocyanidins in different plant species andgrowing conditions (Tonelli et al.,1991; Paolocci et al., 2005). The second example concerns genetic engineering of carrot stem cells to produce astaxanthin. Astaxanthin is a carotenoid produced in few green algae as in Haematococcus lacustris (ex pluvialis), highly appreciated by pharmaceutical and nutritional industries for its high antioxidant capabilities. Here (b) we transformed carrot stem cells with the gene for ?-carotene ketolase from Chlamydomonas reinhardtii: this enzyme converts ?- carotene into astaxanthin in unicellular algae (Perozeni et al., 2020). In the third case ©, we transiently transformed tobacco leaf protoplasts with a construct coding for the human enzyme IDO1. By promoting the degradation of tryptophan to produce kynurenines, molecules capable to modulate the immune system, this enzyme exerts a powerful immunoregulatory action. Therefore, we are currently testing the hypothesis of tobacco cells as a IDO1 biofactory.

Recently, transformation of chloroplast genome has been used for the production of heterologous proteins. We transformed tobacco chloroplasts with two different versions of the storage protein of Phaseolus vulgaris, phaseolin (with or without signal peptide), in which a cysteine residue has been added to its C-terminal region. This modification allows for the formation of inter-chain disulfide bonds, as previously demonstrated in our lab. Our purpose here is to demonstrate the different ability of chloroplast compartments (stroma and thylakoids) in the formation of phaseolin polypeptides held together by disulfide bonds. We observed that the presence of the signal peptide directs phaseolin into the thylakoid compartment, where the protein is able to form disulfide bridges and high molecular weight polymers, which represent about 0.05 % of the total soluble proteins. The formation of phaseolin polymers, not detected in P. vulgaris, could be very interesting for industrial purposes. The chloroplast could be utilized as a platform for the production of a biopolymer that derives from an edible protein. A possible application is the production of biodegradable films.

Hemophilia A is a inherited bleeding disorder caused by deficiency of coagulation factor VIII (FVIII). The FVIII replacement therapy with infusion of plasma-derived or recombinant functional FVIII protein is the current standard treatment for this disease. Transgenic plants are largely used for synthesis and accumulation of foreign proteins as they represent a cost-effective alternative to microbial systems. Our aim is to transform tobacco plants with the cDNA encoding for the B-domain-deleted FVIII (FVIII-BDD). The FVIII-BDD protein extracted from the extracellular media of human cells or Chinese hamster ovary cells exhibits coagulant activity. Because of few data are available in literature about the exact composition of the common drugs for the haemophilia A treatment, some preliminary biochemical experiments have been carried out to characterize the behavior of different FVIII products currently used for the factor replacement therapy. Our data show that different pharmaceuticals contain both the whole chain and the two polypeptides (light chain and heavy chain) generated by the intracellular FVIII proteolysis. The cloning strategy to produce the FVIII in plants requires the insertion of the human gene into a plasmid capable of expressing the gene construct in plant cells. Here we describe the FVIII cloning and the preliminary data obtained by experiments carried out on tobacco protoplasts transiently transformed with the construct encoding the FVIII-BDD protein.

Olive fly (Bactrocera oleae R.) is the most harmful insect pest of olive (Olea europaea L.) which strongly affects fruits and oil production. Despite the expanding economic importance of olive cultivation, up to now, only limited information on plant responses to B. oleae is available. Here we demonstrate that olive fruits respond to B. oleae attack by producing changes in an array of different defensive compounds including phytohormones, volatile organic compounds (VOCs) and defence proteins. B. oleae-infested fruits induced a strong ethylene burst and transcript levels of several putative ethylene responsive transcription factors (ERFs) became significantly up-regulated. Moreover, infested fruits induced significant changes in the levels of 12-oxo-phytodienoic acid (OPDA) and C12 derivatives of the hydroperoxide lyase (HPL). The emission of VOCs was also changed quantitatively and qualitatively in insect-damaged fruits, indicating that B. oleae larval feeding can specifically affect the volatile blend of fruits. Finally, we show that larval infestation maintained high levels of trypsin protease inhibitors in ripe fruits, probably by affecting post-transcriptional mechanisms. Our results provide novel and important information to understand the response of the olive fruit to B. oleae attack; information that can shed light onto potential new strategies to combat this pest.

New selections systems may help to overcome the difficulty of plastid genome transformation in many plant species, allowing to extend the application of this technology. A mutant hemL gene from the photosynthetic cyanobacterium Synechococcus, encoding a gabaculine-insensitive glutamate 1-semialdehyde aminotransferase (GSA), is an efficient selectable marker gene for nuclear transformation of tobacco, alfalfa and durum wheat. Since GSA functions in the plastid, we introduced the mutant hemL gene into the tobacco plastid genome along with the conventional antibiotic resistance aadA gene, in the attempt to develop a new selection system for plastome transformation. Although we were unable to directly regenerate gabaculine resistant transplastomic plants, we demonstrated the functionality of hemL in tobacco plastids by using gabaculine selection in the second and third rounds of in vitro selection that permitted to obtain the homoplasmic state in transgenic plants. Thus, the mutant hemL gene functions as a secondary selection marker in tobacco plastids. Our results encourage further attempts to test gabaculine resistant GSA genes for plastome transformation of crop plants in which gabaculine has stronger regeneration-inhibiting effects with respect to tobacco.

Olive fly (Bactrocera oleae R.) is the most harmful insect pest of olive (Olea europaea L.) which strongly affects fruits and oil production. Despite the expanding economic importance of olive cultivation, up to now, only limited information on plant responses to B. oleae is available. Here, we demonstrate that olive fruits respond to B. oleae attack by producing changes in an array of different defensive compounds including phytohormones, volatile organic compounds (VOCs), and defense proteins. Bactrocera oleae-infested fruits induced a strong ethylene burst and transcript levels of several putative ethylene-responsive transcription factors became significantly upregulated. Moreover, infested fruits induced significant changes in the levels of 12-oxo-phytodienoic acid and C12 derivatives of the hydroperoxide lyase. The emission of VOCs was also changed quantitatively and qualitatively in insect-damaged fruits, indicating that B. oleae larval feeding can specifically affect the volatile blend of fruits. Finally, we show that larval infestation maintained high levels of trypsin protease inhibitors in ripe fruits, probably by affecting post-transcriptional mechanisms. Our results provide novel and important information to understand the response of the olive fruit to B. oleae attack; information that can shed light onto potential new strategies to combat this pest.

High-mobility group box 1 protein (HMGB1) is anuclear component, but extracellularly it servesas a signaling molecule involved in acute andchronic inflammation, for example in sepsisand arthritis. The identification of HMGB1 inhibitorsis therefore of significant experimental andclinical interest. We show that glycyrrhizin, anatural anti-inflammatory and antiviral triterpenein clinical use, inhibits HMGB1 chemoattractantand mitogenic activities, and has aweak inhibitory effect on its intranuclear DNAbindingfunction. NMR and fluorescence studiesindicate that glycyrrhizin binds directly toHMGB1 (Kd 150 mM), interacting with twoshallow concave surfaces formed by the twoarms of both HMG boxes. Our results explainin part the anti-inflammatory properties of glycyrrhizin,and might direct the design of newderivatives with improved HMGB1-bindingproperties.

In the past twenty years, plastids have become attractive targets in the field of plant biotechnology. Plastid transformation is routinely used only in tobacco, thus it is very important for the application of chloroplast engineering to extend the range of species in which this technology can be achieved. Here, we describe the development of a chloroplast transformation system for the sugar beet (Beta vulgaris L. ssp. vulgaris, Sugar Beet Group) by biolistic bombardment of leaf petioles and the production of sugar beet transplastomic plants. Despite being a dicotyledonous plant and the considerable attention it has received due to its importance as an agricultural crop for sugar production in the Northern Hemisphere, sugar beet is considered to be a recalcitrant species with respect to genetic transformation, displaying poor reproducibility and genotype dependency. Moreover, there are no reports of sugar beet chloroplast DNA transformation at present, making this field of research open to exploration. The production of transplastomic sugar beet lines with maternal inheritance of transgenes could solve problems related to outcrossing between genetic modified varieties and conventional varieties or wild relatives. It is well documented that the sugar beet easily inter-crosses with its wild relative, the sea beet [(Beta vulgaris L. ssp. maritima (L.) Arcang.)], or with annual weed beets that occur in the field. Although Beta vulgaris is a biennial crop that is harvested before its reproductive stage and is not expected to flower in the first year, early flowering can occur through occasional vernalization at low spring temperatures. Therefore, unintentional pollination of sea beets or weed beets is possible. The use of chloroplast genetic engineering could drastically reduce the probability of transgenic pollen dispersal. To develop a chloroplast transformation system for this species, different methods for plant regeneration were tested for all the varieties analysed. Homoplasmic plastid-transformed plants of breeding line Z025 were obtained. Transformation was achieved using a vector that targets genes to the rrn16/rps12 intergenic region of the sugar beet plastome, employing the aadA gene as a selectable marker against spectinomycin and the gfp gene for visual screening of plastid transformants. gfp gene transcription and protein expression were shown in transplastomic plants. Detection of GFP in Comassie blue-stained gels suggested high GFP levels. Microscopy revealed GFP fluorescence within the chloroplasts. Our results demonstrate the feasibility of engineering the sugar beet chloroplast genome.

Plants are potentially useful for the production of many therapeutic proteins and several strategies have been exploited to maximize heterologous protein accumulation in the plant cell. Recently, a fusion protein made between the bean storage protein phaseolin and the N-terminal portion of maize prolamin storage protein ?-zein was shown to accumulate in novel ER-derived protein bodies of vegetative tissues, while ?-zein and phaseolin usually accumulates in the ER and in storage vacuoles respectively. The ?-zein fragment, on the basis of the knowledge acquired on zeolin, could be exploited for the production of large amounts of recombinant pharmaceuticals in transgenic plants. In this study we investigate if zeolin could be also expressed in the chloroplast and not only in the ER. In fact, together with others numerous advantages such as high-level of transgene expression, in many crop species chloroplast DNA is not transmitted through pollen thus eliminating the risk of transgene spreading in not transgenic crops or relative wild species. The zeolin gene has been cloned in three plastid expression cassettes. Two of them have been assembled to evaluate the utility of including in the zeolin gene the region that encodes the signal peptide. In the third construct the BiP gene, which encodes an ER chaperone, has been cloned downstream of the zeolin gene. The presence of the signal peptide seems to promote zeolin accumulation in the chloroplast, even though a comparison between the two zeolin sub-cellular localizations shows that zeolin has accumulated in the chloroplast to lower levels than in the ER. Immunoblotting of the transplastomic plant protein shows the considerable presence of phaseolin and ?-zein polypeptides with respect to intact zeolin. This suggests that in the chloroplast a higher amount of zeolin is fragmented in comparison to zeolin accumulated in the ER. Moreover, no protein bodies have been detected in the chloroplast. In order to promote zeolin folding into protein bodies in this organelle, we have integrated the BiP gene together with the zeolin gene in the tobacco plastome but even in this case transplastomic plants has not accumulated zeolin in protein bodies. We hypothesize that the disulfide bonds necessary for zeolin assembly have not been correctly formed in the chloroplast. This can have caused zeolin misfolding and not-stable proteins that reach poor expression levels due to chloroplast proteases activity.

The major seed storage proteins of maize (Zea mays) and bean (Phaseolus vulgaris), zein and phaseolin, accumulate in theendoplasmic reticulum (ER) and in storage vacuoles, respectively.We show here that a chimeric protein composed of phaseolinand 89 amino acids of g-zein, including the repeated and the Pro-rich domains, maintains the main characteristics of wild-typeg-zein: It is insoluble unless its disulfide bonds are reduced and forms ER-located protein bodies. Unlike wild-type phaseolin,the protein, which we called zeolin, accumulates to very high amounts in leaves of transgenic tobacco (Nicotiana tabacum). Arelevant proportion of the ER chaperone BiP is associated with zeolin protein bodies in an ATP-sensitive fashion. Pulse-chaselabeling confirms the high affinity of BiP to insoluble zeolin but indicates that, unlike structurally defective proteins that alsoextensively interact with BiP, zeolin is highly stable. We conclude that the g-zein portion is sufficient to induce the formation ofprotein bodies also when fused to another protein. Because the storage proteins of cereals and legumes nutritionallycomplement each other, zeolin can be used as a starting point to produce nutritionally balanced and highly stable chimericstorage proteins.

Fibroblast growth factor 2 (FGF-2) is a potent factor modulating the activity of many cell types. Its dimerization and binding to high affinity receptors are considered to be necessary steps to induce FGF receptor phosphorylation and signaling activation. A structural analysis was carried out and a region encompassing residues 48-58 of human FGF-2 was identified, as potentially involved in FGF-2 dimerization. A peptide (FREG-48-58) derived from this region strongly and specifically inhibited FGF-2 induced proliferation and migration of primary bovine aorta endothelial cells (BAEC) in vitro, and markedly reduced FGF-2-dependent angiogenesis in two distinct in vivo assays. To further investigate the role of region 48-58, a polyclonal antibody raised against FREG-(48-58) was tested and was found to block FGF-2 action in vitro. Human FGF-2 has three histidine residues, one falling within the region 48-58. Chemical modification of histidine residues blocked FGF-2 activity and FREG-(48-58) inhibitory effect in vitro, indicating that histidine residues, in particular the one within FREG-(48-58) region, play a crucial role in the observed activity. Additional experiments showed that FREG-(48-58) specifically interacted with FGF-2, impaired FGF-2-interaction with itself, with heparin and with FGF receptor 1, and inhibited FGF-2-induced receptor phosphorylation and FGF-2 internalization. These data indicate for the first time that region 48-58 of FGF-2 is a functional domain controlling FGF-2 activity.