Comparative transcriptome and metabolite survey reveal key pathways involved in the control of the chilling injury disorder superficial scald in two apple cultivars, 'Granny Smith' and 'Ladina'.

The low temperature normally applied to prevent fruit decay during the storage of apples, can also triggers the onset of a chilling injury disorder known as superficial scald. In this work, the etiology of this disorder and the mechanism of action of two preventing strategies, such as the application of 1-MCP (1-methylcyclopropene) and storage at low oxygen concentration in 'Granny Smith' and 'Ladina' apple cultivars were investigated. The metabolite assessment highlighted a reorganization of specific metabolites, in particular flavan-3-ols and unsaturated fatty acids, while the genome-wide transcriptomic analysis grouped the DEGs into four functional clusters. The KEGG pathway and GO enrichment analysis, together with the gene-metabolite interactome, showed that the treatment with 1-MCP prevented the development of superficial scald by actively promoting the production of unsaturated fatty acids, especially in 'Granny Smith'. 'Ladina', more susceptible to superficial scald and less responsive to the preventing strategies, was instead characterized by a higher accumulation of very long chain fatty acids. Storage at low oxygen concentration stimulated a higher accumulation of ethanol and acetaldehyde together with the expression of genes involved in anaerobic respiration, such as malate, alcohol dehydrogenase and pyruvate decarboxylase in both cultivars. Low oxygen concentration, likewise 1-MCP, through a direct control on ethylene prevented the onset of superficial scald repressing the expression of PPO, a gene encoding for the polyphenol oxidase enzyme responsible of the oxidation of chlorogenic acid. Moreover, in 'Granny Smith' apple, the expression of three members of the VII subgroups of ERF genes, encoding for elements coordinating the acclimation process to hypoxia in plants was observed. The global RNA-Seq pattern also elucidated a specific transcriptomic signature between the two cultivars, disclosing the effect of the different genetic background in the control of this disorder.

Strategies to produce T-DNA free CRISPRed fruit trees via Agrobacterium tumefaciens stable gene transfer.

Genome editing via CRISPR/Cas9 is a powerful technology, which has been widely applied to improve traits in cereals, vegetables and even fruit trees. For the delivery of CRISPR/Cas9 components into dicotyledonous plants, Agrobacterium tumefaciens mediated gene transfer is still the prevalent method, although editing is often accompanied by the integration of the bacterial T-DNA into the host genome. We assessed two approaches in order to achieve T-DNA excision from the plant genome, minimizing the extent of foreign DNA left behind. The first is based on the Flp/FRT system and the second on Cas9 and synthetic cleavage target sites (CTS) close to T-DNA borders, which are recognized by the sgRNA. Several grapevine and apple lines, transformed with a panel of CRISPR/SpCas9 binary vectors, were regenerated and characterized for T-DNA copy number and for the rate of targeted editing. As detected by an optimized NGS-based sequencing method, trimming at T-DNA borders occurred in 100% of the lines, impairing in most cases the excision. Another observation was the leakage activity of Cas9 which produced pierced and therefore non-functional CTS. Deletions of genomic DNA and presence of filler DNA were also noticed at the junctions between T-DNA and genomic DNA. This study proved that many factors must be considered for designing efficient binary vectors capable of minimizing the presence of exogenous DNA in CRISPRed fruit trees.