These authors contributed equally to this work. SUMMARYLow temperature severely influences potato production as the cultivated potato (Solanum tuberosum) is frost sensitive, however the mechanism underlying the freezing tolerance of the potato is largely unknown. In the present research, we studied the transcriptome and metabolome of the freezing-tolerant wild species Solanum acaule (Aca) and freezing-sensitive cultivated S. tuberosum (Tub) to identify the main pathways and important factors related to freezing tolerance. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation indicated that polyamine and amino acid metabolic pathways were specifically upregulated in Aca under cold treatment. The transcriptome changes detected in Aca were accompanied by the specific accumulation of putrescine, saccharides, amino acids and other metabolites. The combination of transcriptome and metabolome analyses revealed that putrescine exhibited an accumulative pattern in accordance with the expression of the arginine decarboxylase gene ADC1. The primary role of putrescine was further confirmed by analyzing all three polyamines (putrescine, spermidine, and spermine) and the genes encoding the corresponding enzymes in two sets of potato genotypes with distinct freezing tolerance, implying that only putrescine and ADC1 were uniquely enhanced by cold in the freezingtolerant genotypes. The function of putrescine was further analyzed by its exogenous application and the overexpression of SaADC1 in S. tuberosum cv. E3, indicating its important role(s) in cold-acclimated freezing tolerance, which was accompanied with the activation of C-repeat binding factor genes (CBFs). The present research has identified that the ADC1-associated putrescine pathway plays an important role in coldacclimated freezing tolerance of potato, probably by enhancing the expression of CBF genes.

Invertase inhibitors (InvInh) interacted with invertases (Inv) and inhibited their activities involved in reducing sugars (RS) accumulation in cold-stored potato tubers. Understanding their potential contribution to RS accumulation is of both theoretical and practical importance because RS accumulation is a costly postharvest problem for both potato producers and processors. In this study, four genes with significant sequence homology to NtInvInhs were identified from potato and their possible contributions to cold-induced sweetening (CIS) of tubers were investigated together with StInv1, an acid invertase gene previously clarified corresponsive to CIS. Transcripts analysis of these StInvInhs and StInv1 among six potato genotypes with distinct CIS sensitivity indicated that StInvInh2 had a negative power regression to RS increase of the cold-stored tubers while a positive linear regression was obtained with StInv1. The relative expression ratio calculated by StInv1/StInvInh2 performed a very significant correlation to RS accumulation, suggesting a possible interaction between StInv1 and StInvInh2 in response to CIS. The bimolecular fluorescence complementation visualized the interaction between StInv1 and StInvInh2A and with StInvInh2B in both onion epidermal cells and tobacco BY-2 cells and demonstrated that these two inhibitors may be the isoforms of StInvInh2 as the counterparts of StInv1. The recombinant StInvInh2B protein inhibited the activities of soluble acid invertase indicating evidently its inhibitory properties. Our results strongly suggest that the interaction between StInv1 and StInvInh2 may play critical roles in controlling the CIS through posttranslational regulation of StInv1 by StInvInh2 in potato tubers and will provide novel tools and resources for improving CIS tolerance of potatoes.

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HighlightStAmy23, StBAM1 and StBAM9 play distinct roles in potato cold-induced sweetening by preferentially acting on soluble phytoglycogen, soluble starch and starch granules, respectively, in different subcellular locations.