Modulation of the glycoalkaloid biosynthesis pathway in potato (Solanum tuberosum L.) and development of CRISPR/Cas9 methodology for tomato (Solanum lycopersicum L.)

Abstract: Steroidal glycoalkaloids (SGAs) are toxic defence substances present in Solanum species, including the tomato and potato. Concerns about SGA toxicity have long been raised regarding table potato cultivars, and more recently regarding starch potato cultivars, where SGAs negatively affect the use of proteins and fibre from starch by-products. Aiming at reducing the SGA hazard in potato, the present investigation has characterized SGA metabolism in starch and table potato cultivars. Further, using CRISPR/Cas9 technology, a number of gene-edited potato mutants with reduced SGA levels were generated, targeting the transcription factor StGAME9, as well as six key genes acting in SGA biosynthesis.Transcriptomic and metabolomic profiling revealed both differences and similarities regarding the regulation of SGA levels in starch compared to table potato cultivars. Starch potato cultivars exhibited higher basal SGA levels and dry matter content but lower protein levels compared to table potato cultivars. In all seven types of key gene mutants, a significant SGA reduction was observed in leaves, tubers at harvest, and in tubers subjected to two SGA-inducing conditions: wounding and light exposure. The regulatory role of StGAME9 in SGA biosynthesis was investigated, and key SGA-related genes regulated by StGAME9 were identified. The reduction of SGA levels in mutants was stronger when genes in the postcholesterol pathway of SGA biosynthesis were mutated, than for the pre-cholesterol ones. The results also showed that the mutation of each of these seven genes blocked both the basal and induced SGA synthesis. Notably, several mutants displayed close to SGA-free tubers and a relatively normal phenotype and tuber yield. A DNA-free genome editing method with a high mutation rate was established for the cultivated tomato, providing opportunities for extending the insights of SGA biosynthesis in potato to tomato.The generation of SGA-free starch potato cultivars now offers an interesting opportunity for industrial applications, e.g. by transforming starch by-products into economically valuable compounds.

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