Preliminary Research On Regulation Of Vacuolar Acid Invertase Activity By SbSnRK1 In Potato

Potato(Solanum tuberosum L.) is the fourth food crop, with great importance in processing like chips, French fries and so on. To reduce potato sprouting and extend the shelf life after harvest, tubers are often stored in low temperature. However, low temperature stimulates the accumulation of reducing sugars in potato tubers. Upon high-temperature processing, these reducing sugars react with free amino acids, resulting in brown, bitter-tasting products and elevated levels of acrylamide – a potential carcinogen which are unacceptable to consumers. Thus, methods that reduce acrylamide are being sought by the potato processing industry. One effective way is to decrease reducing sugars in cold-stored tubers. We previously reported that a class of invertase inhibitors could specifically inhibit the activity of vacuolar invertase. Besides, one of these inhibitors, St Inv Inh2 B, could bind to a vacuolar invertase Stvac INV1 and Sb Sn RK1. They can form a protein complex composed of Sb Sn RK1, Stvac INV1 and St Inv Inh2 B to subtly regulate the acitivity of Stvac INV1. However, the subcellular location of the protein complex is still unclear. The phosphorylation site in Sb Sn RK1α is less understood. We also do not known if the variation of Sb Sn RK1α transcripts abundance results in the changes of phosphorylation levels, which thus lead to the alteration of VI activity. Besides, the function of Sb Sn RK1β in potato cold-induced sweetening(CIS) has not been addressed. Focusing on these issues, regulation of VI activity by Stvac INV1-St Inv Inh2B-Sb Sn RK1α has been further clarified in present research. The main achievements are as follows:1. To analyze subcellular localization of the protein complex in vivo, Stvac INV1, St Inv Inh2 B, Sb Sn RK1α and Sb Sn RK1β were fused with RFP and the tonoplast marker γTIP was fused with GFP. RFP-Sb Sn RK1α, RFP-Sb Sn RK1β, Stvac INV1-RFP and St Inv Inh2B-RFP were co-expressed with the tonoplast marker γTIP-GFP in vacuoles of N. benthamiana leaves. Results show that Sb Sn RK1α and Sb Sn RK1β are localized onto the tonoplast, the same as for γTIP, while Stvac INV1 is localized in the vacuole. The florescence signal of St Inv Inh2 B was detected both at the membrane and inside the vacuole. These results suggest that Sb Sn RK1 could be a specific form localized onto the tonoplast to confer physiological functions by forming a protein complex with Stcav INV1 and St Inv Inh2 B at the membrane or inside the vacuole of plant cells. To our knowledge, this is the first report to show the localization of potato Sn RK1 onto the tonoplast.2. To identify phosphorylation sites of Sb Sn RK1α, Thr-175 and Ser-176 were mutated to Ala separately and simultaneously. Derived mutants are denoted as Sb Sn RK1αT175A, Sb Sn RK1αS176A and Sb Sn RK1αT175A/S176 A, respectively. The mutated proteins were expressed in E. coli with a GST tag and used in the enzyme activity assay of Stvac INV1. The results show that neither Sb Sn RK1αT175A nor Sb Sn RK1αT175A/S176 A is able to restrict the function of Sb Sn RK1β when GRIK1 is present compared to wild type Sb Sn RK1α. Although Sb Sn RK1αS176A partially represses the function of Sb Sn RK1β, the invertase activity is significantly lower than that of Sb Sn RK1αT175A, demonstrating that Thr-175 is a main phosphorylation site of Sb Sn RK1α. Semi-quantifying phosphorylation signals in western blot analysis with phospho-specific Sn RK1 antibodies reveals that the phosphorylation level of Sb Sn RK1α is higher by 1.6- to 2.4- fold in cold-stored tubers of the two OE lines compared with E3, while this phosphorylation could hardly be detected in the Ri tubers. These results indicate that the phosphorylation intensive of Sb Sn RK1α is likely proportional to its transcripts abundance, which could be causal for alteration of the invertase activity in potato tubers.3. To study the function mechanism of Sb Sn RK1β in potato CIS,Sb Sn RK1β was transformed into potato variety E-potato 3 and AC142 by Agrobacterium-mediated RNA interference and over-expression, respectively. The transgenic lines of Sb Sn RK1β were screened in their transcripts for further analysis. After cold storage, not as roles of Sb Sn RK1β played in the protein complex in vitro,the VI acitivity was decreased slightly in Ri-lines. And no obvious variation of RS content was detected. We speculate that it could be caused by a function complementation of the redundant homologs of Sb Sn RK1β in Ri lines.