| Potato (Solanum tuberosum L.), the fourth important food crop in the world, is vital to global food security. After harvest, potato tubers are often stored at low temperature to prevent sprouting and minimize the disease losses. However, low temperature leads to accumulation of reducing sugars (mainly glucose and fructose) in the tubers known as cold-induced sweetening (CIS). During frying in high temperature, the reducing sugars react with free amino acids and result in unacceptable color change and acrylamide formation in potato chips or French fries, which cause major economic losses and healthy concerns. We previously reported that the vacuolar invertase (VI, one of the most critical enzymes in CIS) can be inhibited by endogenous invertase inhibitor (VIH). However, VI activity regulation is still unclear. In present study, proteins associated with StvacINV1activity were captured from potato and the mode of post-translational regulation of VI activity was further clarified both in vivo and in vitro. The main achievements are as below.1. The protein-protein interaction (PPI) protomics of StvacINVl and StInvInh2B in potato CISTo understand the target proteins of VI and VIH, the two yeast-two hybrid (Y2H) libraries were constructed for potato CIS-resistant wild species S. berthaultii acc. CW2-1and CIS-sensitive cv. AC035, respectively. The StvacINV1and StInvInh2B were employed as the baits for Y2H libraries screening, and two protein-protein interaction (PPI) protomics were obtained after false positive analysis. One contained27potential interaction partners of StvacINV1(24from CW2-1library and3from AC035), and is the other incuded8potential target proteins of StInvInh2B (all from CW2-1library). Noticeably, not only putative invertase inhibitors were captured by StvacINV1, but also the a and β subunits of sucrose non-fermenting-1-related protein kinase1(SnRKl), were selected by both StvacINV1and StInvInh2B. The results suggested that, in addition to the invertase inhibitors, other proteins may also contribute to the VI activity modulation at poat-translational level. 2. Confirmation of StvacINV1-StInvInh2B-SbSnRKl complex in plant and the regulatory mode for VI activityThe StvacINV1-StInvInh2B-SbSnRKlprotein complex was reconfirmed in tobacoo cells using BiFC assay. All proteins of the complex including StvacINV1, StInvInh2B, and a and β subunit of SbSnRK1were individually expressed in vivo and purified proteins were obtained for further analysis. The StvacINV1protein was mixed in each possible combinations with StInvInh2B, SbSnRKla and SbSnRK1β. The residual activity of StvacINV1in these protein mixtures was measured and used to indicate the effects of each protein on the alteration of VI activity. The results demonstrated that inhibitory function of StInvInh2B to StvacINV1was suppressed by SbSnRK1β, and phosphorylation of SbSnRKla counteracted SbSnRK1β roles which led to restore of the function of StInvInh2B and, hence, inactivation of StvacINV1. A subtle regulatory mode of VI activity is established.3. SbSnRK1a plays critical roles in potato CISTo justify if the roles of SbSnRKla played in the protein complex have impacts on potato CIS, the SbSnRK1a gene was transformed into a CIS-sensitive potato variety E-Potato3(E3) by Agrobacterium-mediated over-expression (OE) and RNA interference (Ri). In total,15OE-and8Ri-lines were obtained. Depends on the SbSnRK1a transcripts,5OE-and4-Ri lines were further analyzed in potato CIS. After the cold storage, all the OE tubers had chip color remarkably lighter than wild type E3, whereas the Ri tubers showed darker color. This phenomenon was in accordance with the changes of invertase activity that decreased by83～95%in OE tubers and increased by30-100%in Ri tubers after stored at4℃for30days, suggesting that transcriptional elevation of SbSnRK1a remarkably improved chip color by suppressing the invertase activity. This result provides in-depth understanding of the regulation of invertase cleavage pathway in the process of potato CIS. |
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