RNAi-mediated silencing of endogenous <Emphasis Type="Italic">Vlnv</Emphasis> gene confers stable reduction of cold-induced sweetening in potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L. cv. Désirée)

Potato tubers must be cold-stored to extend their shelf life and maintain an uninterrupted supply chain for food processors. However, a side-effect of low-temperature storage is manifested in terms of cold-induced sweetening (CIS) of potato tubers, which reduces the processing quality and the commercial value of the end-products. RNA interference (RNAi) technology, whereby transgene-derived small interfering RNAs can trigger the homology-based knockdown of cognate host genes and can initiate gene silencing, has been successfully applied in crop improvement through targeted gene knockout in host plants. In the current study, transgenic potato plants (Solanum tuberosum cv. Désirée) were generated, expressing a 300 bp hairpin loop nucleotide sequence targeting the potato vacuolar invertase gene (VInv), under the constitutive Cauliflower mosaic virus 35S promoter. Tubers collected from transgenic lines showed a significant reduction in reducing sugar content after 180 days of cold storage, without showing any measurable off-target effects on plant morphology and tuberization compared to non-transformed control plants. The cold-stored tubers were further assayed for chip color, which showed a fairly light colored quality in the samples originating from RNAi lines. Together with similar effects seen in previously published experiments involving other potato varieties, the Désirée results described here establish the efficacy of using RNAi for the successful reduction of CIS in potato tubers.     

Lipoxygenase Pathway and Membrane Permeability and Composition during Storage of Potato Tubers (Solanum tuberosum L. cv Bintje and Désirée) in Different Conditions

Abstract: Potato tubers ( Solanum tuberosum L. cv Bintje and Désirée) were stored for 12 months under three different storage conditions: 4 °C, 20 °C with sprout inhibitor and 20 °C without sprout inhibitor. Independent of the storage conditions, our results show that the increase of membrane permeability, as revealed by electrolyte leakage, is not correlated with the lipid saturation status. Moreover, there is no simple correlation between cold sweetening and membrane permeability or lipid saturation status. During storage at 20 °C without sprout inhibitor, the increase in membrane permeability is inversely correlated to sucrose accumulation, but this is not the case when tubers were stored with sprout inhibitors. Lipoxygenase (LOX) is often proposed as responsible for peroxidative damage to membrane lipids. The gradual peroxidation resulting in double bond index decrease is regarded as a cause of senescence sweetening. Our results revealed that the role of LOX in aging and senescence of potato tubers is far from clear. LOX activity and gene expression are not correlated with the fatty acids composition of the membrane. Moreover, LOX activity and fatty acid hydroperoxide content are low in older tubers, whatever the storage conditions or the varieties. On the basis of our results, the correlation between sugar accumulation (low temperature and senescence sweetening) and peroxidative damage occurring during storage of potato tubers is discussed.     

Acrylamide in processed potato products: progress made and present status

Concerns related to higher levels of acrylamide in processed carbohydrate-rich foods, especially in fried potato products, are well known. This article provides updates on various aspects of acrylamide in processed potato products including mechanisms of acrylamide formation and health risks due to its intake. Levels of reducing sugars in potatoes are considered as a main factor contributing towards the formation of acrylamide in processed potato products. Useful approaches in lowering the levels of reducing sugars such as use of suitable varieties, storage methods, storage temperature and duration of storage are described and discussed. Importance and practical utility of various steps before and during the processing that can contribute in reducing the final concentration of acrylamide are highlighted. Progress made and present status of potato processing industry in India are part of this article. The article describes varietal improvement and spread of short-term and long-term storage technologies in India and their contribution towards round the year availability of processing-grade potatoes to the processing industries and how all this has helped in achieving reduced levels of acrylamide in chips and French fries. Outcome and implications of cold-induced sweetening tolerance in potatoes are presented along with other management practices and strategies that can lower the acrylamide levels in processed potato products. Future lines of work have been suggested to make the consumption of fried potato products safer.     

Cold sweetening in diploid potato: mapping quantitative trait loci and candidate genes

A candidate gene approach has been used as a first step to identify the molecular basis of quantitative trait variation in potato. Sugar content of tubers upon cold storage was the model trait chosen because the metabolic pathways involved in starch and sugar metabolism are well known and many of the genes have been cloned. Tubers of two F(1) populations of diploid potato grown in six environments were evaluated for sugar content after cold storage. The populations were genotyped with RFLP, AFLP, and candidate gene markers. QTL analysis revealed that QTL for glucose, fructose, and sucrose content were located on all potato chromosomes. Most QTL for glucose content mapped to the same positions as QTL for fructose content. QTL explaining >10% of the variability for reducing sugars were located on linkage groups I, III, VII, VIII, IX, and XI. QTL consistent across populations and/or environments were identified. QTL were linked to genes encoding invertase, sucrose synthase 3, sucrose phosphate synthase, ADP-glucose pyrophosphorylase, sucrose transporter 1, and a putative sucrose sensor. The results suggest that allelic variants of enzymes operating in carbohydrate metabolic pathways contribute to the genetic variation in cold sweetening.     

The response of carbohydrate metabolism in potato tubers to low temperature

This work investigates the possible causes of cold-induced sweetening in potato by examining the impact of low temperature on carbohydrate metabolism in mature tubers. Metabolism in tuber discs was monitored by determining the redistribution of radiolabel following incubation in [U-(14)C]glucose. Estimates of flux based on the specific activity of hexose phosphates established that while incubation at 4 degrees C resulted in an immediate restriction in pathways of carbohydrate oxidation relative to activity at 25 degrees C, there was no corresponding increase in flux to soluble sugars. In contrast, prior storage at low temperature stimulated flux to sugars at both 4 and 25 degrees C. Comparison of (14)CO(2) release from specifically labeled glucose and gluconate fed to tuber discs at 4 and 25 degrees C indicated that flux through glycolysis was preferentially restricted relative to the oxidative pentose phosphate pathway at low temperature, irrespective of prior storage temperature. However, the degree of randomization of label between positions C1 and C6 in the fructosyl moiety of sucrose following metabolism of [1-(13)C]glucose established that there was no preferential inhibition of the recycling of triose phosphates to hexose phosphates at low temperature. These results indicate that sugar accumulation in tubers during storage in the cold is not a direct consequence of a constraint in carbohydrate oxidation, despite preferential restriction of glycolysis at low temperature. It is concluded that the cold lability of enzymes catalyzing the conversion of fructose 6-phosphate to fructose 1,6-bisphosphate is not a major factor in cold-induced sweetening in plants and that this widely held hypothesis should be abandoned.     

Comparative proteomic analysis of cold-induced sweetening in potato (Solanum tuberosum L.) tuber

Cold-induced sweetening is one of the major factors limiting the quality of fried potato products. To understand the mechanisms of protein regulation for cold-induced sweetening in potato tubers, a comparative proteomic approach was used to analyse the differentially expressed proteins both during control (25 °C, 30 days) and cold treatment (4 °C, 30 days) using two-dimensional gel electrophoresis. Quantitative image analyses indicated that there were 25 protein spots with their intensities significantly altered more than twofold. Of these proteins, 9 were up-regulated, 13 were down-regulated, 2 were absent, and 1 was induced in the cold-stored tubers. The MALDI-TOF/TOF MS analyses led to the identification of differentially expressed proteins that are involved in several processes and might work cooperatively to maintain metabolic homeostasis in tubers during low-temperature storage. The preponderance of metabolic proteins reflects the inhibition of starch re-synthesis and the accumulation of sugars in carbon fluxes, linking starch–sugar conversion. The respiration-related proteins suggest the transfer of respiratory activity from aerobic respiration to anaerobic respiration in the cold-stored tubers. The proteins associated with defence appear to protect the tuber cells from low-temperature stress. Some heat shock proteins that act as chaperones also displayed a differential expression pattern, suggesting a potentially important role in cold-stored tubers, although their exact contribution remains to be investigated. The proposed hypothetical model might explain the interaction of these differentially expressed proteins that are associated with cold-induced sweetening in tubers.     

Molecular cloning and sequence comparison of a cDNA encoding α-glucan, water dikinase (GWD) from potato (Solanum tuberosum L.), and analysis of gene expression

Starch phosphorylation is an important biochemical aspect of plant starch metabolism as it influences the overall structure of the starch granule, and a prerequisite for its degradation. There is a growing interest on the isolation and characterization of α-glucan/glucan-like, water dikinases (GWDs) from plants, particularly agriculturally important crops, because GWD is known to catalyze starch phosphorylation both in leaves and different plant storage organs. In the present study, a 4,789-bp full-length cDNA encoding a GWD isoform was isolated from a commercially important Indian potato cultivar, Kufri Chipsona-1 by RT-PCR approach using tuber RNA. The predicted protein consisted of 1,463 amino acids having N-terminal 77-amino acid transit peptide, and 1,386-amino acid mature protein shorter by one amino acid as compared to the other mature GWDs from potato and tomato. The mature GWD showed 98 % sequence identity with the GWD isolated earlier from the potato cv. Desiree. Variations were found at 25 locations representing mostly non-conservative substitutions. The GWD represents a distinct isoform from potato, as revealed by sequence and phylogenetic analyses. Amino acid composition, segment-wise hydrophobic characters, predicted secondary structures were also analyzed and documented in this report. Broadly, the level of GWD expression as analyzed by semi-quantitative RT-PCR approach was found to be nearly uniform both in the mature tubers and leaves from most of the potato cultivars. By immunodetection technique, a band corresponding to ~155 kDa protein was detected only in the tuber protein extracts. The tuber starch-bound phosphorus content data showed minor variations between the potato cultivars.     

Nutrient-encapsulation of potato nodal segments for germplasm exchange and distribution

Nutrient-encapsulation technique using in vitro grown nodal segments was developed as an alternative method for distribution of potato germplasm. The nodal cuttings of two potato genotypes, Kufri Jyoti and Kufri Lauvkar, were encapsulated in calcium-free Murashige and Skoog's (MS) medium containing either 2 or 3 % sodium alginate and 0, 1, 2 or 3 % saccharose. The encapsulated segments were stored in tubes with or without semisolid MS medium, and incubated in the dark at 25 ± °C for 3 to 6 weeks. Presence of saccharose in the beads was found detrimental for regrowth of new shoots. In absence of saccharose, about 82 % and 53 % encapsulated segments initiated regrowth after 3 and 6 weeks of dark storage, respectively, in tubes containing MS medium. Storage in empty tubes deteriorated the encapsulated segments, and depressed shoot formation. For potato germplasm distribution, the encapsulated segments can be transported in small tubes containing semisolid MS medium. This revised version was published online in July 2006 with corrections to the Cover Date.     

Production of Phytophthora infestans oospores in planta and inoculum potential of in vitro produced oospores under temperate highlands and subtropical plains of India

High moisture content of the host tissue ( 88%) and low ambient r.h. (50-54%) favoured oospore formation under controlled environments. It took 14–16 days for oospores to develop; thereafter the number of oospores increased with time and decreased with moisture content of host tissue. High ambient r.h. (> 80%) did not favour oospore formation under field or controlled conditions. Oospore formation was detected in inoculated plants grown in the field when the ambient r.h. declined to 74% and moisture content of host tissue decreased to 83.7–85.6%. It took 8 days (cv. Kufri Chandramukhi) to 13 days (cv. Kufri Jyoti and Kufri Badshah) for oospores to develop. Cultivars also differed in their response to oospore production, cv. Kufri Chandramukhi being more responsive (4800 oospores g⁻¹ f wt) than cv. Kufri Jyoti and Kufri Badshah (1320 and 390 oospores g⁻¹ f wt respectively). Oospores produced in vitro remained viable when buried in soil in the temperate highlands of Himachal Pradesh and sub-tropical plains of Uttar Pradesh, India for more than 150 days, i.e. beginning of the next crop season. The oospores germinated and initiated late blight infection at the base of the stems after 21–30 days of incubation of the potato plants raised in oospore-infested soil. It took 2 days for newly formed oospores to germinate and this delay time increased to 75–77 days after 180-days burial. It took 15 days for their germination (47%) in soil extract as compared to 50 days in sterilised distilled water.     

Effect of inoculation density on potato micropropagation

The in vitro microplant growth of two potato genotypes viz., Kufri Jyoti and Kufri Chandramukhi was affected by inoculation density. After 2 weeks of culture, maximum microshoot length and number of roots/microplant were observed at an inoculation density of five single node cuttings per culture tube. There was difference in response between the genotypes for mean number of nodes/microplant. The number of explants cultured per tube did not significantly change microplant growth produced over 4 weeks of culture.     

Vacuolar invertases in potato (Solanum tuberosum L.): molecular cloning, characterization, sequence comparison, and analysis of gene expression in the cultivars

In plants, vacuolar invertase (β-fructofuranosidase, EC 3.2.1.26) is known to play as a key modulator for hexose accumulation and cell expansion. In this study, two cDNA clones (2,013 and 1,945 bp, with 99 % sequence identity) encoding vacuolar invertase isoforms were isolated from a commercially important Indian potato cultivar, Kufri Chipsona-1 by RT-PCR. The corresponding predicted proteins consisted of 635 amino acids (designated as KC-VIN1, lacking a few amino acids at N-terminus) and 639 amino acids (designated as KC-VIN2), respectively. They showed 99 % identity, and found to vary at several locations with mostly non-conservative substitutions. Multiple sequence alignment of vacuolar invertase homologs covering four Solanaceae family members revealed some notable distinguishing sequence features (signature-type sequences). A consensus sequence was predicted using 45 vacuolar invertase sequences from 27 taxonomically different plant species, and a phylogenetic tree was generated to know the evolutionary relation between them. Hydrophobic characters were predicted, and compared in different plant species. All these data are presented in a comprehensive manner which were not documented in the earlier reports. As a preliminary study, vacuolar invertase expression patterns in the tubers of some Indian potato cultivars were analyzed by semi-quantitative RT-PCR and extractable enzyme assay. In all the potato cultivars, the overall expression level of invertase was found to be considerably higher after storage at low temperature as compared to the freshly harvested tubers.     

Evaluation of different multidimensional LC-MS/MS pipelines for isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analysis of potato tubers in response to cold storage

Cold-induced sweetening in potato tubers is a costly problem for the food industry. To systematically identify the proteins associated with this process, we employed a comparative proteomics approach using isobaric, stable isotope coded labels to compare the proteomes of potato tubers after 0 and 5 months of storage at 5 °C. We evaluated both high pH reverse phase (hpRP) liquid chromatography (LC) and off-gel electrophoresis (OGE) as first dimension fractionation methods followed by nanoLC-MS/MS, using two high performance mass spectrometry platforms (Q-TOF and Orbitrap). We found that hpRP-LC consistently offered better resolution, reduced expression ratio compression, and a more MS-compatible workflow than OGE and consistently yielded more unique peptide/protein identifications and higher sequence coverage with better quantification. In this study, a total of 4463 potato proteins were identified, of which 46 showed differential expressions during potato tuber cold storage. Several key proteins important in controlling starch-sugar conversion, which leads to cold-induced sweetening, as well as other proteins that are potentially involved in this process, were identified. Our results suggest that the hpRP-RP shotgun approach is a feasible and practical workflow for discovering potential protein candidates in plant proteomic analysis.     

Potential Role of Pyrophosphate:Fructose 6-Phosphate Phosphotransferase in Carbohydrate Metabolism of Cold Stored Tubers of Solanum tuberosum cv Bintje

To gain a better understanding of the mechanism of cold induced sweetening, sugar accumulation in potato, Solanum tuberosum cv Bintje, was compared to the maximum activity of inorganic pyrophosphate (PPi):fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90) and the concentration of two regulatory metabolites. Mature tubers accumulated reducing sugars and sucrose at an almost linear rate of 13.4 and 5.2 micromole per day per gram dry weight at 2°C and 4.5 and 1.3 micromole per day per gram dry weight, respectively, at 4°C. During storage at 8°C sugar accumulation was nil. Sugar accumulation was preceded by a lag phase of about 4 days. The accumulation of reducing sugars persisted for at least 4 weeks, whereas sucrose accumulation declined after 2 weeks of storage. The ratio of glucose:fructose changed concomitantly with sugar increase from 65:35 to equimolarity. The maximum activity of PPi:fructose 6-phosphate 1-phosphotransferase was 2.51 and 2.25 units per gram dry weight during storage at 2 and 8°C, respectively. The temperature coefficient of this enzyme from potatoes kept at 2 or 8°C was 2.12 and 2.48, respectively. The endogenous concentration of fructose 2,6-biphosphate increased from 0.15 to 1 nanomole per gram dry weight during storage at 2 and 4°C but remained the same throughout storage at 8°C. After exposure to 2°C an initial increase in the concentration of PPi was observed from 4.0 to 5.6 nanomoles per gram dry weight. Pyrophosphate concentration did not change during storage at 4°C but decreased slightly at 8°C. All observed changes became annulled after transfer of cold stored tubers to 18°C. These data strongly indicate that PPi:fructose 6-phosphate 1-phosphotransferase can be fully operational in cold stored potato tubers and the lack of increase in PPi concentration supports the functioning of this enzyme during sugar accumulation.     

Decreased amount of reducing sugars in transgenic potato tubers and its influence on yield characteristics

This work focuses on the comparison of field characteristics and amounts of reducing sugars in cold-stored tubers of transgenic plants derived from two potato cultivars. The bacterial gene coding for phosphofructokinase under the tuber-specific promoter was used to support the glycolysis in stored tubers. While the tubers from untransformed control plants steadily accumulated reducing sugars during cold storage, the tubers from transformed plants regardless the genotype were characterized by subsequent decrease in the sugar content. After long period of cold storage the greatest reduction in the reducing sugar content was by more than 60 % compared to control. Before the storage, however, the content of reducing sugars was in 80 % of transgenic lines higher than in control ones. The plants evaluated in field trials for their appearance showed any changes in growth characteristics in about 25 % of the transgenic lines. Despite the introduced modification of sugar metabolism the yield of transgenic plants with normal appearance did not differ significantly from the yield of control plants.     

Alleviation of low temperature sweetening in potato by expressing Arabidopsis pyruvate decarboxylase gene and stress-inducible rd29A: A preliminary study

The acceptability of potatoes for processing chips and French fries is largely dependent on the color of the finished product. Most potato cultivars and varieties stored at temperatures below 9–10 °C are subjected to low temperature sweetening (LTS) which result in the production of bitter-tasting, dark colored chips and French fries which are unacceptable to consumers. However, storing tubers at low temperatures (i.e., <10 °C) has many advantages such as lowered weight loss during storage, natural control of sprouting, and reduction/elimination of chemical sprout inhibitors. Our earlier research results on LTS suggested a role for pyruvate decarboxylase (PDC) in LTS-tolerance. In the present study, the role of PDC was examined whereby the potato variety Snowden was transformed with Arabidopsis cold-inducible pyruvate decarboxylase gene 1 (AtPDC1) under the control of promoter rd29A. Two transgenic plants were selected and storage studies were conducted on tubers harvested from one of the transgenic lines grown under green house conditions. Transgenic tubers showed higher Agtron chip color score indicating lighter chip and lower reducing sugar and sucrose concentrations compared to the untransformed tubers during the storage periods studied at 12 °C and 5 °C. These results suggest that overexpression of pyruvate decarboxylase gene resulted in low temperature sweetening tolerance in the transgenic Snowden.