CRISPR/Cas9-Mediated Targeted Mutagenesis of CYP93E2 Modulates the Triterpene Saponin Biosynthesis in Medicago truncatula

In the Medicago genus, triterpene saponins are a group of bioactive compounds extensively studied for their different biological and pharmaceutical properties. In this work, the CRISPR/Cas9-based approach with two single-site guide RNAs was used in Medicago truncatula (barrel medic) to knock-out the CYP93E2 and CYP72A61 genes, which are responsible for the biosynthesis of soyasapogenol B, the most abundant soyasapogenol in Medicago spp. No transgenic plants carrying mutations in the target CYP72A61 gene were recovered while fifty-two putative CYP93E2 mutant plant lines were obtained following Agrobacterium tumefaciens-mediated transformation. Among these, the fifty-one sequenced plant lines give an editing efficiency of 84%. Sequencing revealed that these lines had various mutation patterns at the target sites. Four T0 mutant plant lines were further selected and examined for their sapogenin content and plant growth performance under greenhouse conditions. The results showed that all tested CYP93E2 knock-out mutants did not produce soyasapogenols in the leaves, stems and roots, and diverted the metabolic flux toward the production of valuable hemolytic sapogenins. No adverse influence was observed on the plant morphological features of CYP93E2 mutants under greenhouse conditions. In addition, differential expression of saponin pathway genes was observed in CYP93E2 mutants in comparison to the control. Our results provide new and interesting insights into the application of CRISPR/Cas9 for metabolic engineering of high-value compounds of plant origin and will be useful to investigate the physiological functions of saponins in planta

Identification of DNA methyltransferases and demethylases in Solanum melongena L., and their transcription dynamics during fruit development and after salt and drought stresses

DNA methylation through the activity of cytosine-5-methyltransferases (C5-MTases) and DNA demethylases plays important roles in genome protection as well as in regulating gene expression during plant development and plant response to environmental stresses. In this study, we report on a genome-wide identification of six C5-MTases (SmelMET1, SmelCMT2, SmelCMT3a, SmelCMT3b, SmelDRM2, SmelDRM3) and five demethylases (SmelDemethylase_1, SmelDemethylase_2, SmelDemethylase_3, SmelDemethylase_4, SmelDemethylase_5) in eggplant. Gene structural characteristics, chromosomal localization and phylogenetic analyses are also described. The transcript profiling of both C5-MTases and demethylases was assessed at three stages of fruit development in three eggplant commercial F1 hybrids: i.e. 'Clara', 'Nite Lady' and 'Bella Roma', representative of the eggplant berry phenotypic variation. The trend of activation of C5-MTases and demethylase genes varied in function of the stage of fruit development and was genotype dependent. The transcription pattern of C5MTAses and demethylases was also assessed in leaves of the F1 hybrid 'Nite Lady' subjected to salt and drought stresses. A marked up-regulation and down-regulation of some C5-MTases and demethylases was detected, while others did not vary in their expression profile. Our results suggest a role for both C5-MTases and demethylases during fruit development, as well as in response to abiotic stresses in eggplant, and provide a starting framework for supporting future epigenetic studies in the species

INVESTIGATING THE ROLE OF AN ERF TRANSCRIPTION FACTOR IN MEDIATING STRESS RESPONSE AND TOMATO FRUIT RIPENING AND QUALITY

Tomato (Solanum lycopersicum L.) is one of the world's most consumed vegetables and its consumption has been associated with decreased risk of chronic degenerative diseases. Tomato fruit is an important source of antioxidant compounds such as carotenoids, particularly lycopene, ascorbic acid, vitamin E and phenolic compounds. Fruit ripening is regulated by ethylene. Ethylene biosynthesis and signaling are modulated during fruit development and ripening and are involved in several processes such as antioxidant accumulation and softening that affect fruit quality and shelf-life. To date, several strategies have been implemented in tomato to modulate ripening and enhance tomato fruit quality and shelf-life by regulating the expression of genes involved in ethylene biosynthesis, perception or signaling. Among others, AP2/ERF genes are transcription factors which play key roles in several processes, such as plant development, ethylene response, and pathogen resistance. In tomato fruit, they can act as positive or negative regulator of ripening and of ethylene production. Our goal is to elucidate the functional role of ERF F4 gene (Solyc07g053740) in mediating modulation of the tomato fruit ripening during the plant response to abiotic stress and investigatiing its impact on fruit redox balance and antioxidant accumulation. In particular, the ERF F4 locus was targeted in Microtom by CRISPR/Cas 9 technology to generate knockout tomato plants. One month-old T3 offspring mutant and wild-type plants underwent three level of irrigation, consisting in the complete restitution of water (FWR), restitution of 50% of lost water (HWR) and 30% of water restitution (TWR), respectively. Beside no variation was observed in the lycopene and total carotenoid levels in fruit at the red-ripe stage, edited plants showed increased levels of soluble solid content than wild type plants and responded to drought treatment with a higher increase in their antioxidant capacity. These results suggested the involvement of ERF F4 in modulating ripening associated metabolic processes and fruit redox balance in response to abiotic stresses. Ongoing experiments will further investigate the role of the tomato ERF F4 in regulating the expression of genes involved in controlling the metabolism of antioxidants, particularly glutathione and ascorbate pool

A time-saving and cost-effective method to process alloys by Laser Powder Bed Fusion

Definition of the main process parameters, laser power (P), scanning speed (v), hatching distance (hd) and scanning strategy useful for producing dense samples, is fundamental to develop novel alloy compositions for Laser Powder Bed Fusion (LPBF). The present work has two aims: on one side, to verify the processability of a new AlSi10Mg + 4Cu alloy of mixed powders by LBPF; on the other side, on the basis of the experimental analysis, to define a method for processing new alloys. Producing Single Scan Tracks (SSTs), samples with unidirectional scanning strategy and samples with 67° rotated scanning strategy, the proper P-v-hd combinations were identified reaching a final porosity lower than 1.5%. A scenario of hardness and build-up rate vs. energy density is given, to adopt the main process parameters suitable to maximize mechanical properties or productivity. According to the novel method, P-v-hd combinations can be defined through the production and characterization of SSTs and samples with 67° rotated scanning strategy. Through two production steps dense samples can be then obtained, allowing the development of new compositions saving time and reducing costs related to the powder usage. Keywords: Laser Powder Bed Fusion (LPBF), Single scan tracks (SSTs), Hatching distance (hd), Overlap, Process parameters, Hardness, Build-up rat

Simultaneous CRISPR/Cas9 Editing of Three PPO Genes Reduces Fruit Flesh Browning in Solanum melongena L.

[EN] Polyphenol oxidases (PPOs) catalyze the oxidization of polyphenols, which in turn causes the browning of the eggplant berry flesh after cutting. This has a negative impact on fruit quality for both industrial transformation and fresh consumption. Ten PPO genes (named SmelPPO1-10) were identified in eggplant thanks to the recent availability of a high-quality genome sequence. A CRISPR/Cas9-based mutagenesis approach was applied to knock-out three target PPO genes (SmelPPO4, SmelPPO5, and SmelPPO6), which showed high transcript levels in the fruit after cutting. An optimized transformation protocol for eggplant cotyledons was used to obtain plants in which Cas9 is directed to a conserved region shared by the three PPO genes. The successful editing of the SmelPPO4, SmelPPO5, and SmelPPO6 loci of in vitro regenerated plantlets was confirmed by Illumina deep sequencing of amplicons of the target sites. Besides, deep sequencing of amplicons of the potential off-target loci identified in silico proved the absence of detectable non-specific mutations. The induced mutations were stably inherited in the T-1 and T-2 progeny and were associated with a reduced PPO activity and browning of the berry flesh after cutting. Our results provide the first example of the use of the CRISPR/Cas9 system in eggplant for biotechnological applications and open the way to the development of eggplant genotypes with low flesh browning which maintain a high polyphenol content in the berries.Research was financially supported by the project CRISPR/Cas9-mediated gene knock-out in eggplant financed by Compagnia San Paolo.Maioli, A.; Gianoglio, S.; Moglia, A.; Acquadro, A.; Valentino, D.; Milani, AM.; Prohens Tomás, J.... (2020). Simultaneous CRISPR/Cas9 Editing of Three PPO Genes Reduces Fruit Flesh Browning in Solanum melongena L. 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