Sugar units, not chilling units, control endodormancy duration in plants; potato tuber as a case study

Endodormancy (ED) is a crucial stage in the life cycle of many perennial plants, regulated by genetic and environmental factors. Chilling units, growth regulators, and nutrient supply are considered inducers of ED release, but the mechanism governing ED duration is poorly understood. The potato tuber has been used as a model system to study metabolic processes associated with ED release. Cold-induced sweetening is a well-known response of the tuber to chilling. Here, we found that cold stress induces an increase in sugar units in association with plasmodesmatal closure in the dormant bud cells. Tuber sweetening was associated with shortened ED duration after cooling. Heat exposure also caused sugar unit accumulation followed by faster ED release. A logistic function was developed to predict ED duration based on sugar unit measurements. We discovered that ED release is better correlated with the accumulation of sugar units compared to chilling units. CRISPR/Cas9 knockout of the vacuolar invertase gene (StVInv) induced longer ED, but only in cultivars in which the mutation modified the level of sugar units. Our results suggest that sugar units are better predictors of vegetative bud ED duration than chilling units.

StRAP2.3, an ERF‐VII transcription factor, directly activates StInvInh2 to enhance cold-induced sweetening resistance in potato

Potato invertase inhibitor (StInvInh2) positively regulates cold-induced sweetening (CIS) resistance by inhibiting the activity of vacuolar invertase. The distinct expression patterns of StInvInh2 have been thoroughly characterized in different potato genotypes, but the related CIS ability has not been characterized. The understanding of the regulatory mechanisms that control StInvInh2 transcription is unclear. In this study, we identified an ERF‐VII transcription factor, StRAP2.3, that directly regulates StInvInh2 to positively modulate CIS resistance. Acting as a nuclear-localized transcriptional activator, StRAP2.3 directly binds the ACCGAC cis-element in the promoter region of StInvInh2, enabling promoter activity. Overexpression of StRAP2.3 in CIS-sensitive potato tubers induced StInvInh2 mRNA abundance and increased CIS resistance. In contrast, silencing StRAP2.3 in CIS-resistant potato tubers repressed the expression of StInvInh2 and decreased CIS resistance. We conclude that cold-responsive StInvInh2 is due to the binding of StRAP2.3 to the ACCGAC cis-element in the promoter region of StInvInh2. Overall, these findings indicate that StRAP2.3 directly regulates StInvInh2 to positively modulate CIS resistance, which may provide a strategy to improve the processing quality of potatoes.

Differential DNA methylation in the Vinv promoter region controls Cold Induced Sweetening in potato

Control of potato sprouting is important to ensure constant supply of high-quality potato to the industry. Efficient control of sprouting can be achieved by chemical treatment or cold temperature. Recent bans on anti-sprouting molecules are prompting the use of cold storage in the potato value chain. Unfortunately, storage of potato at low temperatures is associated with cold induced sweetening (CIS) due to the induction of the vacuolar invertase gene under low temperatures. Because CIS is associated with the production of the potentially carcinogenic acrylamide in processed potatoes, concise knowledge on the regulatory mechanisms controlling the CIS-phenotype in potatoes is expected to help pave the way for the production of CIS-resistant potato varieties. Here, we dissect the promoters of the Vacuolar invertase (Vinv) genes from CIS-susceptible and CIS-resistant varieties to investigate their implication in CIS-phenotype determination. Using bisulfite sequencing and CRISPR-dCas9-DRM2-mediated de novo DNA methylation, we show that the CIS-resistant phenotype of Verdi, is in part due to hypermethylation of its Vinv promoter, more specifically in the 1.0-1.7kb region. Those findings open new perspectives to engineer CIS-resistant potatoes by genome and epigenome modifications.