Identification And Profiling Of The Genes Associated With Cold-induced Sweetening In Solanum Berthaultii

In order to prolonging the storage for year-round processing of potato tubers, and reduce the loss caused by microorganism infection, sprout growth, and water loss after harvest, low temperature (around4℃) storage is commonly used. However, the cold-induced sweetening (CIS) characterized by accumulation of reducing sugars in the tubers often occurs. As potato tubers are processed into chips and fries, reducing sugars react with amino acids to generate unacceptable dark-colored, bitter-tasting products in the non-enzymatic Maillard reaction, as well as accumulating acrylamide-a known neurotoxin and suspected carcinogen. CIS has posed a significant challenge to potato industry and is of commercial interest.Studies suggest that the level of CIS varies between potato cultivars, some wild species exhibit high level resistance to CIS. Understanding the mechanism of CIS through identifying the regulative pathways and associated genes should be critical for improving potato processing quality.In present study, two potato species, a CIS-resistant wild type species Solanum berthaultii (accession CW2-1, ber) and a CIS-sensitive cultivator (S. tuberosum) E-potato-3(E3), were employed. Differentially expressed genes responsible to low temperature were classified and the genes involved in CIS were elucidated. The main results are as following.1. Response of potato genotypes with distinct resistance to CIS to low temperature in terms of variation in chip color index and soluble sugar contentsTo determine the effects of storage temperature on chip color and contests of reducing sugars and sucrose, ber and E3tubers were stored at4℃and20℃, and sampled at0,3,5,10,15,20,30,45,60,75and90d, respectively. The results indicated that both ber and E3tubers displayed little variation when stored at20℃. However, dramatic variations were observed when tubers stored at4℃. Generally, the reducing sugars and sucrose contents increased along with the time course although there were some declines at the end of the storage. Comparing between the two potato genotypes, ber had a lower reducing sugar and a higher sucrose contents than E3. Importantly, there was a positive linear relationship between the reducing sugar content and the chip color index, suggesting that the wild potato species ber was more resistant to CIS than E3. 2. Construction of the reverse suppression subtractive hybridization (SSH) libraryIn order to identify the genes associated with CIS, a forward SSH libraries of ber tubers subjected to cold stimulation was constructed previously by Yang (2005). A reverse SSH library was constructed in the present study. ber tubers stored at4℃and20℃for5d were sampled and used as Driver and Tester, respectively. The special expressed sequences were obtained by subtraction and selective PCR amplification, and ligated into the pBlueScript SK (-), then transformed into Escherichia coli DH5a competent cells. The transformed E. coli cells were selected by Amp/X-gal/IPTG and PCR. A total of1920white-single colonies were selected. After PCR screening, a total of1584valid clones (a single amplicon around500bp in length) were contained in the reverse SSH library.3. Identification of differentially expressed (DE) genes responsive to low temperatureA total of3696inserts, including2112clones from the forward library and1584from the reverse library, were amplified by PCR and purified, then qualified to ensure adequate and equal PCR products. The amplicons were printed onto glass slides to produce the microarrays. Total RNAs from ber tubers stored at4℃and20℃for5d were prepared and reverse transcribed separately in presence of Cy5-and Cy3-labeled dUTP. The hybridization was preformed using the labeled cDNAs. Finally,736cold-related clones were identified, of which663were putatively up-regulated and73down-regulated. Then the inserted fragments were sequenced and719clones produced good results. A total of188non-redundant sequences were identified by comparative analyses against the GenBank database, including138down-regulated and50up-regulated genes. One hundred and forty-nine genes of known function were classified into14functional categories. These functional genes were mostly related to cell rescue, defense and virulence, metabolism, energy and protein fate, representing various processes of plant defense against abiotic stresses. These results present an extensive investigation of the DE genes of tubers in response to cold stress, providing a basis in understanding possible mechanisms of CIS. 4. Screening of the genes associated with CIS of potato tubersProfiling of the188DE genes as mentioned above in CIS-resistant ber tubers and in CIS-sensitive E3tubers were further investigated by qRT-PCR, tubers were stored at4℃and sampled at20℃for0,5,15and30d, respectively. The results indicated that most of the genes had nearly the same expression patterns in ber and E3tubers, indicating that tubers with different genotypes shared similar cold responses mechanisms. However, some "special genes", which might determine the degree of CIS, were found differentially regulated in ber and E3tubers. During cold storage, transcripts of C20-3-O14(GWD) and C20-6-K02(xylose isomerase) were suppressed in ber tubers, but they were induced in E3tubers, the transcripts accumulation of the two genes were less in ber tubers than in E3. C20-2-D03(BMY7) and C20-4-C15(InvInh) were cold induced genes in ber and E3tubers, but ber tubers had more transcripts accumulation of InvInh and less BMY7than E3. Transcriptional patterns of C20-6-F06(GAPDH) and C20-3-A14(pyruvate kinase) were first up-regulated and then down-regulated in the cold stored ber and E3tubers. However, both the two genes had higher transcriptional levels in ber tubers than in E3. Comparing to E3tubers, ber might have lower amylolysis level but higer glycolysis level under cold conditions, so the sucrose decomposition might be limited in ber tubers. Together, it was hypothesized that amylolysis, sucrose decomposition and glycolysis pathways might be three key-determinants of CIS. Further investigation of these cold-regulated genes will deepen our understanding of the regulatory mechanisms of potato CIS and direct approaches for the genetic improvement of potato processing quality.