Functions Of Sbtrxh1and Sb14-3-3Genes In Cold-induced Sweetening Of Potato Tubers

Potato(Solanum tuberosum) tubers are often stored in cold (<7℃) to prevent loss from water loss, disease infection and sprouting in processing industries (potato chips, French fries, etc.). However, cold storage leads to accumulation of reducing sugars (RS) in the tubers, known as the cold-induced sweetening (CIS). During frying, interaction between RS the free amino acids results in unsavory and unsightly products, and this reaction could also generate a neurotoxin and potential carcinogen, acrylamide.In this research, two genes, SbTRXhl and Sb14-3-3, which were both up-regulated at the early stage of the cold storage of CIS-resistant potato5. berthaultii (CW2-1) tubers were cloned and characterised. Transcriptional patterns of SbTRXhl and Sb14-3-3during the growth and storage period were analyzed. To elucidate the functions of SbTRXh1and Sbl4-3-3in CIS-regulation, the effects of transcriptional modification of the two genes on the RS accumulation of a CIS-sensitive potato (S. tuberosum cv. E-potato-3, E3) tubers were studied, and the interactive proteins of SbTRXhl and Sb14-3-3of the cold stored CW2-1tubers were identified. The main results were as the follows.1. The5'-and3'-fragments of the SbTRXhl and Sb14-3-3were cloned by RACE approach, splicing of the fragments resulted in two complete sequences. The5'flanking sequences of SbTRXh1and Sb14-3-3from CW2-1and E3genome were also cloned and analyzed. SbTRXhl gene spans664bp containing a369bp ORF. Sequence analysis demonstrates that SbTRXhl is a gene that encodes a new isoform of thioredoxin h1in potato, and it has a characteristic W residual at the19th aa and a catalyze domain of "WCGPC". The gDNA sequences of SbTRXhl gene from both CW2-1and E3genome have two introns and three extrons. Stresses and low temperature response elements are found from their5'flanking sequences. Sb14-3-3gene spans1066bpinclude containing a783bp ORF. It was demonstrated to be a novel member of14-3-3ε in potato. The gDNA sequences of Sb14-3-3gene from both CW2-1and E3genome have five introns and six extrons. Stresses and sugar response elements are found from their5'flanking sequences.2. Transcription of both SbTRXh1and Sb14-3-3in potato plants were developmently dependent with no organic specificity. During storage, both SbTRXhl and Sb14-3-3were unregulated in tubers stored in cold, suggesting that they are involved in the response to low temperature of potato tubers.RS contents in Sb14-3-3-or SbTRXh1-transgenic and wild type E3tubers maintained at a low level during storage at20℃, and increased when tubers were stored at4℃. However, RS accumulation rate in Sb14-3-3-RNAi-or SbTRXh1-RNAi-tubers were dramatically lower than that in wild type E3tubers. In combination with significant linear correlations between RS contents and gene transcription in stored tubers, SbTRXhl and Sb14-3-3were suggestd to be involved in regulation of CIS. To the best of our knowledge, this is the first clarifiaction that SbTRXh1and Sb14-3-3function in potato CIS regulation.A decreased RS accumulation rate in RNAi-transgenic tubers was accompanied by a declined sucrose (Sue) accumulation rate, whereas no statistical significant difference was found between the starch contents of RNAi-transgenic and E3tubers. Additionally, positive linear correlations were established between RS and Sue content, and between Sue content and the gene (Sb14-3-3or SbTRXh1) transcription. Therefore, it could be concluded that accumulation of RS in E3tubers in response to the cold storage may be dominantly from the cleavage of sucrose, and accordingly, a low RS accumulation in the cold stored RNAi-transgenic tubers was a consequence of their less Sue accumulation.In the cold stored SbTRXh1-OE-tubers (overecpression), magnificent abundance of SbTRXh1transcripts was accompanied by a limited accumulation of RS and Sue, suggesting a maximum transcripts threshold of SbTRXh1for CIS regulation of potato tubers.3. Sb14-3-3protein and site-direct mutant of SbTRXh1protein were expressed in Escherichia coli and used for the caption of interactive proteins from stored potato tubers through affinity trapping approach. Identification of Sbl4-3-3and SbTRXh1interactive proteins from CW2-1tubers stored at4℃for10d yielded21and33proteins, respectively. The proteins interacted with SbTRXh1were involved in multi-pathway, including glycolysis/gluconeogenesis, purine biosynthesis, signaling, nitrogen metabolism, flavonoids biosynthesis, protein biosynthesis and folding. The proteins tnteracted with Sb14-3-3were calssified to be involved in many metabolisms, such as glucose6-phosphate transport, protein biosynthesis and folding, purine biosynthesis, signaling, RNA splicing, inositol biosynthesis, ATP biosynthesis, amino acid biosynthesis and pigment biosynthesis. The present results provide with clutes for further investigation of the mechanism by which Sb14-3-3and SbTRXh1acomplish their functions in regulation of potato CIS.