Expression Regulatory Mechanisms Of The Potato Vacuolar Acid Invertase Gene

Potato (Solalnum tuberosum L.) is the fourth most important food crop in the wold and China ranks the first with its potato production. The global consumption of potato is shifting from staple food to value-added processed products. Potato tubers are often stored at low temperature to prevent sprouting and minimize disease losses. However, low temperature leads to accumulation of reducing sugars in potato tubers, a process known as the cold-induced sweetening (CIS). Reducing sugars react with free amino acids during frying for potato chips or French fries, resulting in unacceptable color change and acrylamide formation, which cause major economic and healthy concerns. Potato vacuolar acid invertase that hydrolyzes sucrose into glucose and fructose has been confirmed to play an important role in CIS. StvacINVl, which encods a potato vacuolar acid invertase, is induced by low temperature in potato tubers and is a main contributor to invertase activity. However, its expression regulatory mechanismemains to be elucidated.To disclose the factors that regulating the StvacINVl transcription, the promoter of StvacINV1was cloned and analyzed. Futhermore, the microRNAs (miRNA) in connection with CIS were analyzed by deep sequencing of small RNA libraries and degradomes. The main results are as follows:1. Six potato genotypes with distint CIS resistance were selected for dynamic analysis of sugar accumulation and invertase expression patterns in tubers during one month of low temperature storage. The significant increase of sucrose content was found to occur earlier than the raise of reducing sugar content in all the genotypes tested. The activity of soluble acid invertase increased accompanied with sugar accumulation, and the transcripts of StvacINV1increased rapidly in tubers exposed to cold treatment. However, no distinct StvacINVl expression pattern could be clarified between the CIS-resistant and the CIS-sensitive genotypes.2. The5’-flanking sequence of StvacINV1was cloned and the cis-acting elements were predicted. Histochemical assay showed that the StvacINVl promoter governed β-glucuronidase (GUS) expression in potato leaves, stems, roots and tubers. Quantitative analysis of GUS expression suggested that the activity of StvacINVl promoter was suppressed by sucrose, glucose, fructose and cold, while it was enhanced by indole-3-acetic acid (IAA) and giberellic acid (GA3). Further deletion analysis clarified that the promoter regions from-118to-551,-551to-1021, and-1021to-1521were responsive to stimuli of sucrose/glucose, GA3and IAA, respectively. The events of reduction in the promoter activity under low temperature implied other regulatory mechanisms for StvacINV1expression, such as post-trnascriptional regulation.3. Two small RNA and two degradomes libraries were constructed from the potato (10908-06) tubers stored at20℃or4℃for deep sequencing. A total of53known miRNAs,60novel miRNAs and70miRNA*s were identified by small RNA sequencing, and70miRNA-targeted genes were predicted by degradome sequencing. The expression level of36miRNAs/miRNA*s showed significant changes in response to cold. Twenty-four miRNAs/miRNA*s and55targets were selected for the expression profiling. The results showed that12miRNAs/miiRNA*s were differencially expressed between20℃and4℃, and between CIS-resistant and the CIS-sensitive genotypes. Among them,11targets were matched by six miRNA/miRNA*, including three genes whose expressions were significantly different between the temperature treatments and between the potato genotypes. Of these three genes, a gene encoding APETALA2is the target of miR172, and the other two genes are the target of miR396a-3p. miR172was induced by2d-cold-storage in the tubers of10908-06along with a down-regulation of its target, indicating the expression of the APETALA2gene is possibly modulated by miR172. Since no miRNAs were identified to match the StvacINV1gene, and the aboundance of StvacINV1mRNAs was not significantly related to CIS, present results suggest that the StvacINV1activity may be essential for potato CIS. This conclusion reinforces the finding that a post-translational regulation of StvacINVl plays key roles in the process of potato CIS. However, the identified miRNA/miRNA*s, as well as their target genes, may provide new clues for approaching a full understanding of the regulatory mechanisms of potato CIS.