| The potato (Solanum tuberosum), rich in starch, is the fourth food crop and plays important roles in food security of the world. The global consumption of potato is shifting from staple food to value-added processed products. Of the processed category French fries and chips dominate the market. The advantages of being able to process potato tubers directly into chips or fries from cold storage (below 10℃) include less shrinkage, retention of dry matter, decreased disease loss, extended processing period, and the elimination of the need for dormancy-prolonging chemicals. Unfortunately, under the cold conditions, starch is partially converted into soluble sugar, a phenomenon known as cold-induced sweetening (CIS). By the Maillard reaction, the accumulated hexoses (glucose and fructose) react with free amino acid which negatively affects the processing quality. The acid invertase (AI), the enzyme catalyzes sucrose into glucose and fructose, is known to considerably influence the reducing sugar (RS) content in cold-stored potatoes.Acid invertase gene family and their putative inhibitors of potato were systemically analysed in present research. The key acid invertase and its inhibitor which were responsive for CIS were identified. In addition, we partially explained the melocular mechanism for CIS in sucrose metabolism pathway. The main results obtained are as following:1. Six acid invertase genes including 4 cell wall invertase and 2 vacuolar invertase genes were identified from potato DNA database. The nucleotide and amino acid sequences of acid invertase gene family share high similarity. To understand the potential function of each member, a comparative expression profiling was performed using the technique of real-time quantitative reverse transcriptase-polymerase chain reaction (Real-time qRT-PCR). Transcript levels of 6 acid invertase genes were analyzed in various organs of potato plants at flowing-stage each showed a unique expression pattern. This analysis was also applied to the cold-stored tubers (7℃for 2 months and 7℃for 2 months followed by reconditioning at 21℃for 3 weeks) of 6 potato genotypes with diverse CIS resistances. The results revealed that only StvacINV1 showed response to the cold stress and this gene was selected for the further research.2. Potato putative Kunize-type invertase inhibitor St-inh and tobacco invertase inhibitor Ntlnvlnh2 genes were expressed in potato cultivar E-potato 3 (E3), respectively. The results showed that the RS accumulicaton was inhibited and sucrose accumulation was elevated due to acid invertase activities been inhibited in the transgentic plants. However, starch content was not obviously changed. The results suggested that these two genes affected the metabolism of sucose in cold-stored potato tubers, giving a potatential of their contributions to improve the processing quality. The results also showed a better impact of Ntlnvlnh2 on the chip color than St-Inh by comparision between their transgenic tubers.3. Four genes with significant sequence homology to Ntlnvlnhs were identified from potato and their possible contributions to CIS of tubers were investigated together with Stlnvl (StvacINVl). The transcripts analysis of these genes in 6 potato genotypes with distinct CIS sensitivity indicated that Stlnvlnh2 had a negative power correlation to the RS content of the cold-stored tubers, while a positive linear correlation was obtained with StInvl, implying that Stlnvlnh2 and Stlnvl may be the major genes involved in the CIS process of potato tubers. Further analysis showed a better correlation between the relative expression ratio (Stlnvl/Stlnvlnh2) and the RS content, suggesting a possible interaction between StInv1 and StInvInh2 in the CIS. This speculation was confirmed by the bimolecular fluorescence complementation of StInv1 and StInvInh2A and StInvInh2B in both onion epidermal cells and tobacco BY-2 cells, demonstrating that the interaction of StInv1 and StInvInh2 may contribute to the variation of RS content of potato tubers among different genotypes. Since similar fluorescence complementation was observed between StInv1 and StInvInh2A and between StInv1 and StInvInh2B, StInvInh2A and StInvInh2B may be the isoforms of StInvInh2. Futhermore, the recombinant StInvInh2B protein inhibited the activities of soluble acid invertase which confirmed evidently its inhibitory properties.Therefore, our results strongly suggest that the CIS of potato tubers may be regulated by the transcription and post-tranlation levels of acid invertase and its inhibitors. The interaction between StInvl and Stlnvlnh2 with posttranslational mechanism may play critical roles in controlling the CIS of potato tubers through regulating the activity of StInvl by its inhibitor protein StInvInh2. Present research will throw a light on approaching new strategy for improving potato CIS resistance.
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