An alternative pathway to plant cold tolerance in the absence of vacuolar invertase activity.

To cope with cold stress, plants have developed antioxidation strategies combined with osmoprotection by sugars. Potato tuber, a swollen stem, exposure to cold stress induces starch degradation and sucrose synthesis. Vacuolar acid invertase (VInv) activity is a significant part of the cold-induced sweetening (CIS) response, by rapidly cleaving sucrose into hexoses, and increasing osmoprotection. To discover alternative plant tissue pathways for coping with cold stress, we produced VInv-knockout lines in two cultivars. Genome editing of VInv in 'Désirée' and 'Brooke' was done using stable and transient expression of CRISPR/Cas9 components, respectively. After storage at 4°C, sugar analysis indicated that the knockout lines showed low level of CIS and maintained low acid invertase activity in storage. Surprisingly, the tuber parenchyma of vinv lines exhibited significantly reduced lipid peroxidation and reduced H 2 O 2 levels. Furthermore, exposing the whole-plant of vinv lines to cold stress, without irrigation, showed normal vigor, in contrast to wilting of the WT plants. Transcriptome analysis of vinv lines revealed upregulation of osmoprotectant, and ethylene-related genes during cold temperature exposure. Accordingly, higher expression of antioxidant-related genes was detected after exposure to short and long cold storage. Sugars measurement showed an elevation of an alternative pathway in the absence of VInv activity, raising the raffinose pathway with increasing levels of myo-inositol content as a cold-tolerance response.