Anthocyanins are valuable water-soluble plant pigments. They accumulate in the vacuole of specialized cells and play a crucial role in pigmentation of flowers and fruits, pollinators attraction, plant-pathogen interaction, protection against ultraviolet (UV) light, and modulation of reactive oxygen species (ROS)-signaling both in reproductive and in vegetative tissues (Brunetti et al., 2013). As plant-derived molecules, anthocyanins are naturally present in the human diet where they are predominantly represented as glycosides, in a multitude of fruits and vegetables, among which berries and grapes have the highest content (Bognar et al., 2013). Anthocyanins act as health-promoting and chronic-diseases-preventing molecules, due to antioxidant, anti-inflammatory, anti-proliferative and anti-neurodegenerative functions (Blesso, 2019; Krga and Milenkovic, 2019; Tian et al., 2019; Bendokas et al., 2020; Kalt et al., 2020). Due to these properties, anthocyanins have increasing applications in the food sector and there is also growing interested in the design of food crops with improved levels and composition of these antioxidant nutraceuticals. In particular, the research involved in the definition of plants intended for ‘agrospace’ applications, will have to tackle the issue to provide fresh and healthy food for space crews in the context of a harmful ionizing irradiated environment, and to cope with cultivation areas subjected to possible biotic contaminations, as well (Amalfitano et al., 2020; Bijlani et al., 2021). Agrospace crops are, therefore, candidates for the application of technologies aiming to improve both their content in antioxidant nutraceuticals and resistance to biotic and abiotic stresses (Zabel et al., 2015, 2016; Massa et al., 2016; Khodadad et al., 2020).

Tomato (Solanum lycopersicum L.) is a rich source of bioactive molecules such as carotenoids (in particular, lycopene), polyphenols and flavonoids, ascorbic acid, and other vitamins (Gerszberg et al., 2015; Martí et al., 2016). However, anthocyanins are poorly accumulated in cultivated tomatoes and even the fruits of cultivar harboring natural mutants for the Abg (Aubergine), Aft (Anthocyanin fruit), and Atv (Atroviolaceum) loci, only contain these molecules in the peel (Mes et al., 2008; Gonzali et al., 2009; Povero et al., 2011). Accumulation in fruit flesh and other organs upon genetic engineering indicates that tomatoes can be manipulated to this end (Zhang et al., 2014; Lloyd et al., 2017). By breeding, tomato lines were generated that combine the dominant Atv allele with Aft or Abg. These lines accumulated up to 0.1% (in fresh weight) of the anthocyanin petunidin-3-(p-coumaryl)-rutinoside-5-glucoside in the fruit epidermis (Mes et al., 2008; Povero et al., 2011). Gene and pathway engineering are powerful approaches to enhance the biosynthesis of anthocyanins in plants, and they have been successfully applied in food staples (Garg et al., 2018). Transcription factors, regulating the expression of structural biosynthetic genes, control the activity of the anthocyanin pathway in all plant species (Gonzalez et al., 2008). In particular, proteins belonging to specific clades of R2R3-MYB, bHLH, WDR, and WRKY have been shown to regulate anthocyanins biosynthesis combined in the MBWW transcription complex, as shown for a multitude of plant species among which tomato (Ramsay and Glover, 2005; Zhang et al., 2014; Gao et al., 2018). The combined expression of a MYB and bHLH regulators of the anthocyanin pathway from Antirrhinum majus (Delila and Rosea1, respectively) under a fruit-specific promoter, resulted in the production of anthocyanins in fruits peel and flesh of tomato (Butelli et al., 2008). Studies have demonstrated that pathway engineering approaches implying the sole use of MYB factors belonging to the SG6 clade are sufficient to restore the biosynthesis of anthocyanins by promotion of the transcription of their bHLH partners and, therefore, by reconstituting the MBWW (Mehrtens et al., 2005; Takos et al., 2006; Zhang et al., 2019). The tomato ANT1 gene encodes a MYB transcription factor belonging to the SG6 clade, highly homologous to the Antirrhinum Rosea. It has been demonstrated that ectopic expression of ANT1 from a tomato wild relative (S. chilense), induces purple spotting on the epidermis of tomatoes (Mathews et al., 2003; Schreiber et al., 2012). The 35S promoter-driven expression of either the Solanum lycopersicum ANT1 or AN2 (another SG6 MYB), has been shown to induce anthocyanins production in the flesh and peel of the fruit and different organs of tomato plants (Kiferle et al., 2015). Upon overexpression of SlAN2, together with anthocyanins accumulation in fruits, flower organs, and vegetative parts, an enhancement of the emission of volatile molecules contributing to the aroma of fruits was found, as well (Jian et al., 2019). In addition, SlAN2 has been related to the variation of levels of specialized metabolites other than anthocyanins, and of fruit softening (Meng et al., 2015). These findings seem to confirm that MYB transcription factors of the SG6 clade can regulate various, sometimes unrelated, processes in tomatoes, as well (Stracke et al., 2001; Zimmermann et al., 2004; Zhang et al., 2019). Therefore, a SG6 MYB-based approach may be considered suitable to affect multiple pathways in tomatoes.

The Anthocyanin4 gene of Petunia hybrida (PhAN4) is a SG6 member of a small family of genes encoding very similar MYBs phylogenetically related to the snapdragon AmROSEA, the tomato SlANT1, and other anthocyanin-regulating MYBs from a multitude of plant species. All these petunia MYBs are involved in the induction of anthocyanins accumulation in different plant parts and response to different stimuli (Povero, 2011). In the present study, we performed Agrobacterium rhizogenes-mediated transfer of a construct for the expression of PhAN4 into the miniature tomato genotype MicroTom to generate hairy root cultures (HRCs). HRCs were intended as a testbed for whole plant engineering strategies able to improve traits for space cultivation. We previously reported about improved in vivo response to space-mimicking conditions (i.e., static magnetic fields and X and gamma rays) of PhAN4-engineered HRC (Villani et al., 2017; Desiderio et al., 2019), confirming that this plant-based expression system, used over the last 30 years to produce various specialized metabolites and recombinant proteins of pharmaceutical value (Gutierrez-Valdes et al., 2020; Häkkinen et al., 2020), is useful in studies on the adaptation of plants to extraterrestrial conditions, as well. Recently, HRCs served in several plant species as handier and faster biotechnology tools, compared to whole plant transformation, to gain biological insights in gene function, spatial and temporal gene expression studies, and signaling pathways in plant cell response to a changing environment (Ron et al., 2014).

Anthocyanin biosynthesis engineering in HRCs was reported in a few species (Sharma et al., 2013; Li et al., 2016; Thwe et al., 2016; Hou et al., 2017), and, to date, never in tomatoes. In this study, PhAN4 gene expression resulted in anthocyanins accumulation in tomato HRCs. Transcript profiling showed that several genes encoding enzymes and transcription factors involved in anthocyanins biosynthesis were upregulated. Interestingly, also genes correlated to cell response to biotic and abiotic stress, including redox stimuli, resulted in transcriptionally upregulated.

In addition, we report about the antioxidant properties and diminished generation of ROS in HRCs expressing PhAN4 exposed to ionizing gamma radiation. PhAN4 HRCs were endowed with a ninefold enhanced antioxidant capacity per se compared to controls by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Furthermore, ROS accumulation was counteracted after gamma radiation, as shown by Electron Spin Resonance (ESR) Spectroscopy. Both UV-VIS spectra and photoluminescence analysis demonstrated that polyphenols content and stability of soluble protein folding were not significantly affected by high dose gamma irradiation in PhAN4-engineered HRCs compared to control.

In conclusion, MicroTom HRCs represented a simplified model that allowed to rapidly test PhAN4 expression effects on tomato cells, possibly opening the way to the application of the strategy to the engineering of whole plants intended for cultivation in harsh environments like future space outposts.

Anthocyanins are well known for their antioxidant and health-protection properties. Tomato has been already subjected to genetic manipulation for improved levels and composition of these high-value compounds (Schauer et al., 2006; Klee and Tieman, 2013; Giovannoni, 2018; Wang et al., 2019). Nutraceutical improvement of tomato is expected to provide a nourishing food source for future long-term manned space missions such as NASA’s “Artemis” lunar exploration program. The Artemis initiative includes sending a suite of new technology demonstrators to establish a sustained human presence by 2028 (DeZwart and Henderson, 2021). Low-power systems to grow plants will have the role to provide fresh and nutritious food to supplement astronauts’ diet and provide psychological benefits. The experiments conducted in the VEGGIE module onboard the International Space Station over the last few years have pioneered this scenario (Wolff et al., 2014; Fu et al., 2016; Massa et al., 2016; Zabel et al., 2016; Imhof et al., 2018).

The space environment, totally unusual for plants, can affect their metabolic processes and, consequently, growth, due to high-energy ionizing radiation, microgravity, magnetic field, and ultra-vacuum (Williams et al., 2004; Lotito and Frei, 2006; Moghaddam et al., 2011; Thamaphat et al., 2015; van Hoeck et al., 2017). Among abiotic stresses that plants will have to cope with in extraterrestrial environments artificially adapted to space agriculture, pro-oxidant stimuli are, indeed, the most prominent, due to cosmic ionizing radiation. Acting directly and indirectly to delay oxidative damage, enzymatic players, and metabolites activating endogenous antioxidant defense systems may help plant growth in the space environment. ROS attack plant cells causing loss of their structure and function (Vandenhove et al., 2010; Moghaddam et al., 2011; Marcu et al., 2013a; Vardhan and Shukla, 2017; Gudkov et al., 2019), rapidly reacting with almost all structural and functional organic molecules in the plant cells and causing irreversible oxidative damage on DNA, lipids, and proteins (Scandalios, 2002; Sewelam et al., 2016). The issue of counteracting overproduction of free radicals generated by harmful ionizing radiation is crucial for human survival in space outposts, as well, and opens the way to the ideal ‘anti-oxidant space fresh food.’ Indeed, the oral intake of clinically tested chemical radioprotectants (i.e., thiols, aminothiols, thiadiazoles, and benzothiazoles) is limited due to toxicity (Copp et al., 2013). Therefore, there is great interest in the development of natural antioxidants possibly produced by plants and introduced with the diet (Gómez et al., 2021). Together with the above-mentioned abiotic ‘macro-stresses,’ plants intended for space agriculture will have to cope with a plethora of abiotic ‘micro-stresses’ related to the different cultivation environment/systems that will be adopted. Furthermore, plants will have to share with humans future crewed space habitats (spaceflights, planetary outposts, and life-support systems) where stringent microbial quality requirements may not be achieved (Amalfitano et al., 2020).

Plants have an intrinsic potential for adaptation as the heritage of ancestors that survived in the harsh initial terrestrial environments (de Vries and Archibald, 2018). The ‘design’ of plant ideotypes suitable to these environments may be achieved by further refinement of existing biochemical and physiological features, such as specialized metabolites, that plants use to survive stress. Novel genotypes boosting biosynthetic pathways for the production of specialized metabolites can be generated by manipulation of their regulators. Among regulators, there are MYB transcription factors, known for their contribution to the increased complexity of land plants and to be regulators of plant responses to the environment (Lloyd et al., 2017; Gao et al., 2018; Kashyap et al., 2020; Shi and Panthee, 2020). Members of this large family of regulators are key players in the modulation, among others, of the biosynthesis of flavonoids, like anthocyanins, in different plant parts and response to all kinds of stimuli (Ambawat et al., 2013; Liu et al., 2015; Roy, 2016). MicroTom is extremely compact, early yielding, and would be suitable for agriculture in the confined area of a crewed space module. Here, we described the ectopic expression of the anthocyanin R2R3-MYB regulator PhAN4 from Petunia hybrida in MicroTom hairy roots as a testbed for future engineered whole plants. As known, anthocyanin production is regulated by R2R3-MYB, bHLH, WDR, and WRKY cooperation (MBWW transcriptional complex) (Spelt et al., 2000; Koes et al., 2005). In this complex, the R2R3-MYB transcription factor is probably the limiting factor, in that the WDR and bHLH proteins are thought to be constitutively expressed (Zhang et al., 2013, 2014). When specific MYB proteins are expressed in plants, this induces anthocyanin accumulation. Since some specific MYB transcription factors can function in regulating not only the biosynthesis of anthocyanins but also other traits (Meng et al., 2015; Vardhan and Shukla, 2017; Jian et al., 2019), we investigated if PhAN4 may affect, together with accumulation of anthocyanins, features associated with improved traits related to survival in harsh and confined environments such as future space outposts.

Differently from the wealth of literature available on whole transgenic plants and cell culture systems, data concerning anthocyanins production are limited for hairy roots. To date, no data are available on anthocyanin accumulation in tomato hairy roots by metabolic engineering to make comparisons with our work. Although not comparable to levels achieved in transgenic plants (Butelli et al., 2008; Povero et al., 2011; Kiferle et al., 2015), accumulation of anthocyanins occurred in MicroTom HRCs upon PhAN4 transformation, as expected due to the nature of the transgene. Importantly, no significant variation of transformed HRCs growth rates was found compared to controls. This gives a clue about the non-detrimental activity of PhAN4 toward primary metabolism in this system.

In accordance with literature related to transgenic tomato plants (Butelli et al., 2008; Su et al., 2016; Jian et al., 2019), mass spectrometry allowed us to identify glycosylated and acylated delphinidin and petunidin as the most abundant anthocyanins in PhAN4 HRCs. Interestingly, two additional anthocyanins, unusual in tomato, pelargonidin-3-glucoside, and malvidin-3-O-(4”’coumaroyl)-rutinose-5-O-glucose, were detected, as well. Malvidin and Pelargonidin derivatives have already been detected in Del and Ros1 and LC and C1 tomato plants, respectively (Bovy et al., 2002; Su et al., 2016). Interestingly, pelargonidin plays a major role in reducing genotoxic stress induced by environmental toxicants in plants (Khandelwal and Abraham, 2014). In addition, rather than other anthocyanins, pelargonidin-3-glucoside, that was accumulated in engineered MicroTom HRCs, has high bioavailability, being absorbed in an intact form into the gastrointestinal wall, undergoing first-pass metabolism and entering the systemic circulation as 4-hydroxybenzoic acid, a stable metabolite that is considered one of the main players of the systemic health effects of anthocyanins (Fang, 2014). In order to achieve a better understanding of PhAN4 in the tomato hairy root phenylpropanoid pathway, we measured 14 phenolic acids and derivatives and 29 flavonoids: notably, a reduction in the content of some members of the former, and a massive increase in most of the compounds in the latter were observed, thus proving PhAN4 is able to trigger the metabolic flux at both flavonoid and anthocyanin levels.

Data on the accumulation of anthocyanins and phenylpropanoids obtained by mass spectrometry was supported by transcriptome-wide RNA-seq analysis. RNA-seq, in complex, depicted a reprogramming oriented not only to anthocyanin accumulation, as expected, but also to positive regulation of cell response to biotic and abiotic stress and, possibly, to fruit quality-related traits.

Expression of PhAN4 increased the transcript levels of almost all of the genes encoding enzymes required for anthocyanin biosynthesis with the exception of PAL, C4H, 4CL, F3′H, and 5UFGT. To facilitate comparisons, transcriptomic research on tomato plants for anthocyanins enrichment has been summarized in Table 2. CHS and CHI had been already found to be upregulated by overexpression of SlAN2 in tomatoes, but not by ectopic expression of Del/Ros that, in turn, were able to upregulate F3H (Butelli et al., 2008; Jian et al., 2019). DFR had been already shown to be upregulated in the stem and leaf of tomato plants overexpressing SlAN2 and SlAN1 R2R3-MYBs (Kiferle et al., 2015). Interestingly, despite FLS was not found among upregulated DEGs in PhAN4 HRCs, LDOX, a known bi-functional enzyme being able not only the conversion of leucoanthocyanidins in anthocyanidins but also to catalyze the formation of flavonols, resulted to be differentially upregulated in PhAN4 HRCs. This may explain the accumulation of quercetin, kaempferol derivatives, and myricetin together with anthocyanidins. The flavonol biosynthetic activity of the upregulated LDOX is counterbalanced by the upregulated DFR. In tomatoes, DFR has a substantial preference for dihydromyricetin, which can be also derived by dihydrokaempferol and dihydroquercetin by F3′5′H activity. Interestingly, DilFl a tomato homolog of cytochrome b5 of P. hybrida, where it is essential for full activity of F3′5′H, was found to be upregulated in PhAN4 HRCs. These findings may explain the differential accumulation of delphinidin and petunidin compared to pelargonidin, malvidin, and cyanidin (the latter was not detected at all). Among late biosynthetic genes, upregulation of ANS had been already shown upon SlAN2 over-expression and Del/Ros expression in tomatoes. OMT and AAT transferases have already been described to be targets of a MYB in tomatoes, as well (Zhang et al., 2019). Their upregulation in PhAN4 HRCs, together with that of GST, PH3, OMT, 3UFGT, RT, and AAT may explain the accumulation of the glycosylated and acylated forms of anthocyanidins. In particular, the function of the upregulated PH3 (Solyc10g084380.1.1) in PhAN4 HRC is rather to be associated with anthocyanin biosynthesis boosting than with acidification of pH, as tomato lacks PH1 and PH5 (i.e., the genes encoding the vacuolar P-ATPases which cooperate with PH3 and PH4 in determining flower color by hyperacidification of petal cell vacuoles in Petunia). Nevertheless, preliminary evaluation of pH of PhAN4 and control HRC homogenates (Verweij et al., 2008) does not allow to exclude that the acidification function may occur (Supplementary Figure 5), given that pH differences between PhAN4 and control HRC seem to be in accordance with variations recorded between vacuolar P-ATPases-defective mutants and wild type Petunia hybrida (Faraco et al., 2014). The upregulation of anthocyanin biosynthesis-relevant transcription factors further expands the influence of PhAN4 MYB in tomato gene expression regulation. Among those, the upregulation of SlAN1, SlAN2, and WD40 are coherent with already proposed models of anthocyanin biosynthesis regulation (Koes et al., 2005; Liu et al., 2018).

Interestingly, the PhAN4-related transcriptomic enhancement was found to involve more than 30 genes specifically related to response to biotic/abiotic and oxidative stress, as well. Indeed, GOE analysis was associated with proteinase inhibitors, beta-glucosidases, glucanases, glycosylases, xylan acetylases, resistance proteins, and other classes of enzymes that are players of defense against pathogens and pests and of response to abiotic stresses specifically associated with wounding, cold, heat, drought, hypoxia, and to UV exposure in tomato (Fan et al., 2020). Among those, the upregulated beta-glucosidases, together with the dirigent proteins and the monoterpenoid and sesquiterpenoid synthases that were found in the present work, are involved in the formation of required intermediates for cell wall lignification. The positive regulation of trichome birefringence-like protein mediating xylan acetylation also is another tool in possible protection against environmental stresses among which there is cold and excess of minerals in the soil. Acetylation of wall polymers is, indeed, vital for plant growth and adaptation to various environments, and is required for the structural integrity of the leaf surface exerting a global impact on plant stress responses (Gao et al., 2017). In relation to the positive regulation of beta-1,3-glucanases, beta-glucosidases, and xylan acetylases, together with expansions, it has to be added that these genes are related to fruit softening or improved emission of volatiles, as well (Tzin et al., 2015; Li P. et al., 2020). In this sense, the positive regulation of chlorophyll a/b binding protein (that is normally upregulated during fruit ripening) and of one gene related to volatile compounds biosynthesis (chorismate mutase) was found in PhAN4 HRCs. At the same time, negative regulation of other genes, which down expression is involved in the extended shelf life of fruits, was found. The evaluation of these aspects might be important in view of the development/design of whole transgenic tomato plants in relation to the quality traits of fruits.

One player of ROS detoxification and regulator of the redox signaling network of tomato, plastidial thioredoxin Y2, was found to be highly upregulated, as well. Moreover, the analysis demonstrated the downregulation of violaxanthin de-epoxidase (VDE) and carotenoid isomerase (CRTISO), two genes involved in carotenoid biosynthesis. Despite being involved in the photo-inhibition of the PSII, suppression of VDE can induce, at the same time, an accumulation of fucoxanthin that functions as an efficient anti-oxidant in anoxia conditions (Mikami and Hosokawa, 2013). The downregulation of carotenoid isomerase was demonstrated to induce an accumulation of zeta-carotene and cis-prolycopene in tomato fruits (Isaacson et al., 2002; Pinheiro et al., 2019), both elevating and modifying carotenoid profiles toward more bioavailable forms compared to wild-type (Cooperstone et al., 2015). Therefore, the ROS counteracting potential of PhAN4 HRC material may be related not only to anthocyanins but also to other accumulating anti-oxidant specialized metabolites.

In view of the future development of natural anti-oxidants produced by engineered plants and possibly administered with the diet, and to define whether the significantly higher DPPH antioxidant capacity of specialized metabolites accumulating in PhAN4 HRCs may be efficiently maintained after ionizing radiation, we exposed lyophilized biomass from AN4-1 HRC to high dose ⁶⁰Co gamma radiation. Gamma rays are a component of cosmic ionizing radiation and induce the formation of free radical species (i.e., paramagnetic species). When carried out in an air atmosphere, gamma rays induce the formation of ROS, such as superoxide, peroxide, and hydroxyl radicals, that are responsible for several oxidative processes in biological systems (Marcu et al., 2013b; Schreurs et al., 2016). Gamma rays are perfect ROS inducers both directly through water radiolysis, and indirectly via the activation of a broad range of signaling processes (e.g., damages to the mitochondria or cell microenvironment) (Riley, 1994; Azzam et al., 2012; Buttarelli et al., 2019). ESR Spectroscopy was used as a sensitive tool to identify the entity of the paramagnetic species generated in the HCRs samples after gamma irradiation (Aleksieva et al., 2009; Hawkins and Davies, 2014; D’Errico et al., 2018). ESR analyses revealed a significant difference in the overall amounts of radical species accumulated in the two HRC molecular backgrounds. At the same absorbed dose, control generates a very intense peroxyl radical signal, while a considerably lower level of the singlet intensity is generated in AN4-1. This result reveals that the molecular set formed upon PhAN4 expression provides very effective free radical scavengers efficiently counteracting oxidative stress upon gamma radiation.

The UV-VIS analysis allowed us to confirm the results obtained by metabolomic data and to characterize the effect of ionizing radiation on the antioxidant compounds. Gamma irradiation, via releasing free radicals in solution, may alter these plant constituents (Lateef and Al-Nimer, 2009; Lalande et al., 2019). Compared to the substantially unchanged spectra of AN4-1, the absorbance decrease of the 320 nm peak (i.e., flavonols, hydroxycinnamic acids, tannins, flavonols, and anthocyanins associated to caffeic and coumaric acids) controls upon irradiation, suggests that the anthocyanins accumulated in PhAN4 HRCs are particularly stable to gamma high absorbed dose and probably have protective effects on other biomolecules. Indirectly, this result shows that these molecules may give a major contribution to the ROS buffering capacity under radiation shown by ESR and to the overall antioxidant potential. As a confirmation of the possible significance of this result in vivo, it was reported that, even at higher absorbed doses than that used in this work, X rays (e.g., another type of ionizing radiation) leave the level of flavonoids in an aqueous solution unchanged due to the radiolysis-mediated formation of depsides that, in addition, maintain good anti-oxidant properties, as well (Kozlowski et al., 2007; Lateef and Al-Nimer, 2009).

For proteins to be functional within a cell requires coordinated folding processes to obtain a correct 3D shape. Disruptions to protein folding, that can occur in space due to ionizing radiation, can have profound biological implications for all organisms, including plant cells, leading to dysfunctions (Blanco et al., 2018; Lalande et al., 2019). For this reason, investigations were conducted through photoluminescence analysis on HRCs’ total soluble protein extracts. When a protein is exposed to the wavelength of 280 nm, mainly the tryptophan and tyrosine residues get excited, which would reflect upon its tertiary structure (Yang H. et al., 2015). The maximum and emission peak position reflects upon secondary, tertiary, and quaternary structures (Hilaire et al., 2017). Moreover, the extent of protein oxidation is measured by determining the loss of specific tryptophan fluorescence. The emission spectra of HRCs showed an overall decrease in maximum emission intensities only in the case of irradiated controls, revealing that the structure of soluble proteins is partially lost upon irradiation in those samples. This result demonstrates a low capacity of proteins of control HRCs to counteract unfolding, binding to hydrophobic pockets, and aggregation, witnessed by the loss of fluorescence due to the burial of tryptophan residues after radiation. In particular, the Δ% photoluminescence intensity/mass units after and before irradiation indicated that soluble proteins from control undergo a doubled oxidative stress compared to AN4-1 samples. Being polyphenols emitting at 280 and 320 nm extracted by PBS together with total soluble proteins (anthocyanins are not efficiently extracted, Supplementary Figure 4), the contribution of these classes of molecules may be hypothesized in stabilizing proteins, as proofs of the interaction of anthocyanins with proteins seem to suggest (Sui et al., 2018). Possible additional contributions of enzymatic players to mitigation of misfolding and aggregation in vivo may be the object of future studies.

In conclusion, plant biotechnology methods may thus be exploited for the generation of plants capable of dealing with harsh conditions such as those typical of space outposts. MicroTom HRC allowed to rapidly test PhAN4 expression effects on tomato cells, possibly opening the way to apply to the engineering of whole plants able to perform in a suitable and predictable manner in those environments.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://dataview.ncbi.nlm.nih.gov/object/PRJNA794337?reviewer=estprpllasrt3f3libdoij8rco.

SM planned and designed the project, undertook tomato transformation experiments, PCR screening and maintenance of HRCs, and sample preparation for subsequent analysis, as well as wrote the manuscript. RP handled maintenance of HRCs, contributed to sample preparation and transcriptomic data retrieving and interpretation, and contributed to the writing of the manuscript and the preparation of figures. AB performed sequencing read mapping, identification of DEGs and functional annotation, and enrichment pathway analysis of DEGs. HP undertook cDNA library construction and sequencing for transcriptomic analysis. MB contributed to the identification of DEGs and functional annotation of genes. AC and IS performed gamma irradiation experiments, ESR, UV-VIS, and photoluminescence analysis and contributed to writing the manuscript. GD and OD performed MS analysis, anthocyanin identification/quantification, and contributed to writing the manuscript. FP conducted the qPCR analysis. AD and PD performed the Trolox assay and the quantification of total polyphenol content and contributed to writing the manuscript. FQ, RK, and CS assembled and kindly provided the PhAN4 constructs used in this work. ElB contributed to the maintenance of HRCs. EuB reviewed and contributed to the writing of the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.