Biofortified tomato plants as a test bed for space agriculture

Abstract

Future long-term space missions will focus to the solar system exploration with the Moon and Mars as the leading goals. As already planned by the Artemis program led by NASA and supported by the main Space agencies, Lunar and Martian missions will be characterized by extended duration. In future deep-space manned missions, when resources or supplies are limited, novel methods for production of edible biomass and recycling of resources are expected to provide solutions for long-term crew survival. During these missions, higher plants will take a crucial role as they produce oxygen, reduce carbon dioxide and recycle water, as well as being a fundamental fresh food source for a nutritionally balanced crew diet, actively contributing to the crew psychological well-being, as well. Isolation, limited resources, microgravity and ionizing radiation (IR) are considered the main hazards of spaceflights due to their capacity to generate cell metabolic stress. IR are the most prominent pro-oxidant stimuli that plants have to cope with because they could both directly damage the DNA structure or generate free radicals through the radiolysis process. The issue of counteracting overproduction of free radicals generated by harmful IR is crucial not only for plant development but also for human survival in space outposts and, in the latter sense, opens the way to the ideal ‘antioxidant space fresh food’. Therefore, there is great interest in the development of biofortified plants accumulating natural antioxidants that may be introduced with the diet of astronauts, as well. Among secondary metabolites that give protection against environmental challenges, anthocyanins are well-known for their potent anti-oxidant properties and, recently, they have been proposed as a component of antioxidant formulations to be administered during spaceflight to maintain space explorers’ health. Indeed, efforts to figure out how plants might perform predictably upon extraterrestrial environment and, possibly, to support life in the confined and unhealthy environment of a space outpost, are needed. This PhD thesis pioneers the development of plant ideotypes aimed at space application through the design of tomato plants (cv. MicroTom) expressing the R2R3-MYB transcription factor PhAN4 from Petunia hybrida. Tomato (Solanum lycopersicum L.) is one of the most cultivated vegetable worldwide and its fruits are the largest dietary source of several nutraceutical metabolites. In particular, the dwarf cultivar MicroTom is one of the crops that meets many of the specific requirements for space cultivation. Despite the richness in valuable compounds known for their nutraceutical effects, accumulation of flavonoids in tomato fruits is sub-optimal and, with some exception, anthocyanins are not present. To elucidate if the PhAN4 gene was able to restore the anthocyanin production, a simplified MicroTom-derived hairy root cultures (HRCs) model was established and then characterized using a transcriptome-wide RNA-seq approach and mass spectroscopy analysis. The constitutive expression of PhAN4 in the HRCs model resulted in modulation of gene expression related the anthocyanin biosynthetic pathway together with modulation of gene expression related to abiotic, biotic and redox stimuli. In addition, AN4 HRCs showed significantly higher capability to counteract ROS accumulation and protein misfolding upon a high pro-oxidant stimulus (i.e., gamma rays) compared to controls. These results paved the way to whole plant engineering. Transgenic lines harboring a single copy of the transgene inserted respectively in the chromosome 8 and 12 were characterized for the PhAN4 ectopic expression. Both homozygous AN4-M and AN4-P plants, and, in particular, the hemizygous AN4-P2 line, showed a purple pigmentation throughout their structure, from leaves and leaf veins, to stem and roots, to anthers and ovary. The phenotypic characterization revealed that the engineered genotypes were unchanged in life cycle as compared to the untransformed control and retained the dwarf phenotype (even smaller) while maintaining the same yield. Total anthocyanin content evaluation and DPPH assay demonstrated the accumulation of anthocyanins in fruits and the increase in the antioxidant capacity compared to wild type. Engineered fruits were also evaluated by Electron Spin Resonance Spectroscopy (ESR) and Photoluminescence analysis. These analyses showed lower accumulation of reactive oxygen species and decreased protein folding damage compared to wild type, upon an ex vivo pro-oxidant stimulus (i.e., 2 kGy of gamma rays). To study how the engineered MicroTom could possibly perform upon ionizing radiation, we exposed seeds and plants at two different developmental stages (30 and 45 DAS) at doses that plants would possibly experience during a long-term space mission (0 Gy, 0.5 Gy, 5 Gy or 30 Gy of gamma rays originated from a 60Co source). These experiments allowed us to tackle the lack of knowledge regarding the effects of gamma rays on fully developed tomato plants and on their offspring. Plants response to gamma rays were evaluated following two approaches: biometric parameters were monitored to detect changes occurred at both phenotypic and phenological level and multiparametric fluorescence-based indices were used to indirectly measure changes occurred at physiological level in a non-destructive manner. Results showed that irradiation of the dry seeds did not hamper germination and seed-to-seed cycle, demonstrating that, at this phenological stage, seeds are more resistant to ionizing radiation due to their structure. Experiments conducted on plants irradiated at 30 and 45 DAS revealed that plants can tackle gamma rays doses until 5 Gy through adjustments at the physiological level. Indeed, low doses of ionizing radiation (0.5 and 5 Gy) delivered at both seed stage and fully developed plants do not impair plants growth and, in some cases, can led to beneficial traits (e.g., smaller size or increased number of seeds). Conversely, between 5 and 30 Gy there is a limiting dose that compromise the plant development and the production of fruits and viable seeds. Interestingly, fluorescence-based indices (i.e., SFR, NBI, FLAV and ANTH) allowed us to detect a bi-phasic response to IR in plants irradiated at 30 DAS and a less pronounced response in plants irradiated at 45 DAS. This ‘lack of response’ could be due to an age-related resistance to stress, highlighting the different effect of gamma rays delivered to plant at different developmental stages. Irradiation of plants at 30 DAS also affected different parameters on offspring, with changes that may be ascribed to epigenetic modifications and altered frequency of transitional recombination. Lastly, gamma rays, independently from the genotype and developmental stage, did not caused alteration in the efficiency of photosynthetic apparatus. In conclusion, among the three genotypes considered, AN4-M plants, maybe to the favourable genomic asset and the favourable content of anthocyanins, demonstrated a high capacity to overcome the stress of gamma rays exposure. All together, the information obtained contribute to advance our understanding of space effects on plants, opening the way to the development of novel plant ideotypes with enhanced favourable traits suitable for future long-term space mission.