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1.  Induction of vacuolar invertase inhibitor mRNA in potato tubers contributes to cold-induced sweetening resistance and includes spliced hybrid mRNA variants 
Journal of Experimental Botany  2011;62(10):3519-3534.
Cold storage of tubers of potato (Solanum tuberosum L.) compromises tuber quality in many cultivars by the accumulation of hexose sugars in a process called cold-induced sweetening. This is caused by the breakdown of starch to sucrose, which is cleaved to glucose and fructose by vacuolar acid invertase. During processing of affected tubers, the high temperatures involved in baking and frying cause the Maillard reaction between reducing sugars and free amino acids, resulting in the accumulation of acrylamide. cDNA clones with deduced proteins homologous to known invertase inhibitors were isolated and the two most abundant forms, termed INH1 and INH2, were shown to possess apoplastic and vacuolar localization, respectively. The INH2 gene showed developmentally regulated alternative splicing, so, in addition to the INH2α transcript encoding the full-length protein, two hybrid mRNAs (INH2β*A and INH2β*B) that encoded deduced vacuolar invertase inhibitors with divergent C-termini were detected, the result of mRNA splicing of an upstream region of INH2 to a downstream region of INH1. Hybrid RNAs are common in animals, where they may add to the diversity of the proteome, but are rarely described in plants. During cold storage, INH2α and the hybrid INH2β mRNAs accumulated to higher abundance in cultivars resistant to cold-induced sweetening than in susceptible cultivars. Increased amounts of invertase inhibitor may contribute to the suppression of acid invertase activity and prevent cleavage of sucrose. Evidence for increased RNA splicing activity was detected in several resistant lines, a mechanism that in some circumstances may generate a range of proteins with additional functional capacity to aid adaptability.
PMCID: PMC3130176  PMID: 21393382
Cold-induced sweetening; cold stress; hybrid RNA; invertase inhibitor; RNA splicing; Solanum tuberosum
2.  Novel candidate genes influencing natural variation in potato tuber cold sweetening identified by comparative proteomics and association mapping 
BMC Plant Biology  2013;13:113.
Higher plants evolved various strategies to adapt to chilling conditions. Among other transcriptional and metabolic responses to cold temperatures plants accumulate a range of solutes including sugars. The accumulation of the reducing sugars glucose and fructose in mature potato tubers during exposure to cold temperatures is referred to as cold induced sweetening (CIS). The molecular basis of CIS in potato tubers is of interest not only in basic research on plant adaptation to environmental stress but also in applied research, since high amounts of reducing sugars affect negatively the quality of processed food products such as potato chips. CIS-tolerance varies considerably among potato cultivars. Our objective was to identify by an unbiased approach genes and cellular processes influencing natural variation of tuber sugar content before and during cold storage in potato cultivars used in breeding programs. We compared by two-dimensional polyacrylamide gel electrophoresis the tuber proteomes of cultivars highly diverse for CIS. DNA polymorphisms in genomic sequences encoding differentially expressed proteins were tested for association with tuber starch content, starch yield and processing quality.
Pronounced natural variation of CIS was detected in tubers of a population of 40 tetraploid potato cultivars. Significant differences in protein expression were detected between CIS-tolerant and CIS-sensitive cultivars before the onset as well as during cold storage. Identifiable differential proteins corresponded to protease inhibitors, patatins, heat shock proteins, lipoxygenase, phospholipase A1 and leucine aminopeptidase (Lap). Association mapping based on single nucleotide polymorphisms supported a role of Lap in the natural variation of the quantitative traits tuber starch and sugar content.
The combination of comparative proteomics and association genetics led to the discovery of novel candidate genes for influencing the natural variation of quantitative traits in potato tubers. One such gene was a leucine aminopeptidase not considered so far to play a role in starch sugar interconversion. Novel SNP’s diagnostic for increased tuber starch content, starch yield and chip quality were identified, which are useful for selecting improved potato processing cultivars.
PMCID: PMC3750364  PMID: 23919263
3.  Validation of candidate gene markers for marker-assisted selection of potato cultivars with improved tuber quality 
Tuber yield, starch content, starch yield and chip color are complex traits that are important for industrial uses and food processing of potato. Chip color depends on the quantity of reducing sugars glucose and fructose in the tubers, which are generated by starch degradation. Reducing sugars accumulate when tubers are stored at low temperatures. Early and efficient selection of cultivars with superior yield, starch yield and chip color is hampered by the fact that reliable phenotypic selection requires multiple year and location trials. Application of DNA-based markers early in the breeding cycle, which are diagnostic for superior alleles of genes that control natural variation of tuber quality, will reduce the number of clones to be evaluated in field trials. Association mapping using genes functional in carbohydrate metabolism as markers has discovered alleles of invertases and starch phosphorylases that are associated with tuber quality traits. Here, we report on new DNA variants at loci encoding ADP-glucose pyrophosphorylase and the invertase Pain-1, which are associated with positive or negative effect with chip color, tuber starch content and starch yield. Marker-assisted selection (MAS) and marker validation were performed in tetraploid breeding populations, using various combinations of 11 allele-specific markers associated with tuber quality traits. To facilitate MAS, user-friendly PCR assays were developed for specific candidate gene alleles. In a multi-parental population of advanced breeding clones, genotypes were selected for having different combinations of five positive and the corresponding negative marker alleles. Genotypes combining five positive marker alleles performed on average better than genotypes with four negative alleles and one positive allele. When tested individually, seven of eight markers showed an effect on at least one quality trait. The direction of effect was as expected. Combinations of two to three marker alleles were identified that significantly improved average chip quality after cold storage and tuber starch content. In F1 progeny of a single-cross combination, MAS with six markers did not give the expected result. Reasons and implications for MAS in potato are discussed.
Electronic supplementary material
The online version of this article (doi:10.1007/s00122-012-2035-z) contains supplementary material, which is available to authorized users.
PMCID: PMC3607734  PMID: 23299900
4.  Vacuolar Invertase Gene Silencing in Potato (Solanum tuberosum L.) Improves Processing Quality by Decreasing the Frequency of Sugar-End Defects 
PLoS ONE  2014;9(4):e93381.
Sugar-end defect is a tuber quality disorder and persistent problem for the French fry processing industry that causes unacceptable darkening of one end of French fries. This defect appears when environmental stress during tuber growth increases post-harvest vacuolar acid invertase activity at one end of the tuber. Reducing sugars produced by invertase form dark-colored Maillard reaction products during frying. Acrylamide is another Maillard reaction product formed from reducing sugars and acrylamide consumption has raised health concerns worldwide. Vacuolar invertase gene (VInv) expression was suppressed in cultivars Russet Burbank and Ranger Russet using RNA interference to determine if this approach could control sugar-end defect formation. Acid invertase activity and reducing sugar content decreased at both ends of tubers. Sugar-end defects and acrylamide in fried potato strips were strongly reduced in multiple transgenic potato lines. Thus vacuolar invertase silencing can minimize a long-standing French fry quality problem while providing consumers with attractive products that reduce health concerns related to dietary acrylamide.
PMCID: PMC3973568  PMID: 24695527
5.  Heterologous microarray experiments allow the identification of the early events associated with potato tuber cold sweetening 
BMC Genomics  2008;9:176.
Since its discovery more than 100 years ago, potato (Solanum tuberosum) tuber cold-induced sweetening (CIS) has been extensively investigated. Several carbohydrate-associated genes would seem to be involved in the process. However, many uncertainties still exist, as the relative contribution of each gene to the process is often unclear, possibly as the consequence of the heterogeneity of experimental systems. Some enzymes associated with CIS, such as β-amylases and invertases, have still to be identified at a sequence level. In addition, little is known about the early events that trigger CIS and on the involvement/association with CIS of genes different from carbohydrate-associated genes. Many of these uncertainties could be resolved by profiling experiments, but no GeneChip is available for the potato, and the production of the potato cDNA spotted array (TIGR) has recently been discontinued. In order to obtain an overall picture of early transcriptional events associated with CIS, we investigated whether the commercially-available tomato Affymetrix GeneChip could be used to identify which potato cold-responsive gene family members should be further studied in detail by Real-Time (RT)-PCR (qPCR).
A tomato-potato Global Match File was generated for the interpretation of various aspects of the heterologous dataset, including the retrieval of best matching potato counterparts and annotation, and the establishment of a core set of highly homologous genes. Several cold-responsive genes were identified, and their expression pattern was studied in detail by qPCR over 26 days. We detected biphasic behaviour of mRNA accumulation for carbohydrate-associated genes and our combined GeneChip-qPCR data identified, at a sequence level, enzymatic activities such as β-amylases and invertases previously reported as being involved in CIS. The GeneChip data also unveiled important processes accompanying CIS, such as the induction of redox- and ethylene-associated genes.
Our Global Match File strategy proved critical for accurately interpretating heterologous datasets, and suggests that similar approaches may be fruitful for other species. Transcript profiling of early events associated with CIS revealed a complex network of events involving sugars, redox and hormone signalling which may be either linked serially or act in parallel. The identification, at a sequence level, of various enzymes long known as having a role in CIS provides molecular tools for further understanding the phenomenon.
PMCID: PMC2358903  PMID: 18416834
6.  Analysis of subcellular metabolite levels of potato tubers (Solanum tuberosum) displaying alterations in cellular or extracellular sucrose metabolism 
Metabolomics  2008;4(2):161-170.
The expression of a heterologous invertase in potato tubers (Solanum tuberosum) in either the cytosol or apoplast leads to a decrease in total sucrose content and to an increase in glucose. Depending on the targeting of the enzyme different changes in phenotype and metabolism of the tubers occur: the cytosolic invertase expressing tubers show an increase in the glycolytic flux, accumulation of amino acids and organic acids, and the appearance of novel disaccharides; however, these changes are not observed when the enzyme is expressed in the apoplast [Roessner et al. (2001). Plant Cell, 13, 11-29]. The analysis of these lines raised several questions concerning the regulation of compartmentation of metabolites in potato tubers. In the current study we addressed these questions by performing comparative subcellular metabolite profiling. We demonstrate that: (i) hexoses accumulate in the vacuole independently of their site of production, but that the cytosolic invertase expression led to a strong increase in the cytosolic glucose concentration and decrease in cytosolic sucrose, whereas these effects were more moderate in the apoplastic expressors; (ii) three out of four of the novel compounds found in the cytosolic overexpressors accumulate in the same compartment; (iii) despite changes in absolute cellular content the subcellular distribution of amino acids was invariant in the invertase overexpressing tubers. These results are discussed in the context of current models of the compartmentation of primary metabolism in heterotrophic plant tissues.
Electronic supplementary material
The online version of this article (doi:10.1007/s11306-008-0107-5) contains supplementary material, which is available to authorized users.
PMCID: PMC2758360  PMID: 19816536
Compartmentation; Plant heterotrophic carbon metabolism; Maltose biosynthesis; Metabolite profiling
7.  Natural DNA variation at candidate loci is associated with potato chip color, tuber starch content, yield and starch yield 
Complex characters of plants such as starch and sugar content of seeds, fruits, tubers and roots are controlled by multiple genetic and environmental factors. Understanding their molecular basis will facilitate diagnosis and combination of superior alleles in crop improvement programs (“precision breeding”). Association genetics based on candidate genes is one approach toward this goal. Tetraploid potato varieties and breeding clones related by descent were evaluated for 2  years for chip quality before and after cold storage, tuber starch content, yield and starch yield. Chip quality is inversely correlated with tuber sugar content. A total of 36 loci on 11 potato chromosomes were evaluated for natural DNA variation in 243 individuals. These loci included microsatellites and genes coding for enzymes that function in carbohydrate metabolism or transport (candidate loci). The markers were used to analyze population structure and were tested for association with the tuber quality traits. Highly significant and robust associations of markers with 1–4 traits were identified. Most frequent were associations with chip quality and tuber starch content. Alleles increasing tuber starch content improved chip quality and vice versa. With two exceptions, the most significant and robust associations (q < 0.01) were observed with DNA variants in genes encoding enzymes that function in starch and sugar metabolism or transport. Comparing linkage and linkage disequilibrium between loci provided evidence for the existence of large haplotype blocks in the breeding materials analyzed.
Electronic supplementary material
The online version of this article (doi:10.1007/s00122-008-0746-y) contains supplementary material, which is available to authorized users.
PMCID: PMC2358939  PMID: 18379755
8.  Low-acrylamide French fries and potato chips 
Plant Biotechnology Journal  2008;6(8):843-853.
Acrylamide is produced in starchy foods that are baked, roasted or fried at high temperatures. Concerns about the potential health issues associated with the dietary intake of this reactive compound led us to reduce the accumulation of asparagine, one of its main precursors, in the tubers of potato (Solanum tuberosum). This metabolic change was accomplished by silencing two asparagine synthetase genes through ‘all-native DNA’ transformation. Glasshouse-grown tubers of the transformed intragenic plants contained up to 20-fold reduced levels of free asparagine. This metabolic change coincided with a small increase in the formation of glutamine and did not affect tuber shape or yield. Heat-processed products derived from the low-asparagine tubers were also indistinguishable from their untransformed counterparts in terms of sensory characteristics. However, both French fries and potato chips accumulated as little as 5% of the acrylamide present in wild-type controls. Given the important role of processed potato products in the modern Western diet, a replacement of current varieties with intragenic potatoes could reduce the average daily intake of acrylamide by almost one-third.
PMCID: PMC2607532  PMID: 18662372
acrylamide; intragenic; plant biotechnology; potato
9.  Potato processing scenario in India: Industrial constraints, future projections, challenges ahead and remedies — A review 
Indian potato (Solanum tuberosum L.) processing industry has emerged fast due to economic liberalization coupled with growing urbanization, expanding market options and development of indegenous processing varieties. India’s first potato processing varieties ‘Kufri Chipsona-1’ and ‘Kufri Chipsona-2’ were developed in 1998, followed by an improved processing variety ‘Kufri Chipsona-3’ in 2005 for the Indian plains and first chipping variety ‘Kufri Himsona’ for the hills. These varieties have >21% tuber dry matter content, contain low reducing sugars (<0.1% on fresh wt) and are most suitable for producing chips, French fries and dehydrated products. The availability of these varieties and standardization of storage techniques for processing potatoes at 10–12°C with sprout suppressant isopropyl N-(3-chlorophenyl) carbamate have revolutionized the processing scenario within a short span of 10 years. Currently about 4% of total potato produce is being processed in organized and unorganized sector. Potato processing industry mainly comprises 4 segments: potato chips, French fries, potato flakes/powder and other processed products. However, potato chips still continue to be the most popular processed product. The major challenge facing the industries lies in arranging round the year supply of processing varieties at reasonable price for their uninterrupted operation, besides several others which have been discussed at length and addressed with concrete solutions.
PMCID: PMC3550973  PMID: 23572617
Potato; Tuber quality; Indian processing varieties; Products; Storage
10.  Natural variation of potato allene oxide synthase 2 causes differential levels of jasmonates and pathogen resistance in Arabidopsis 
Planta  2008;228(2):293-306.
Natural variation of plant pathogen resistance is often quantitative. This type of resistance can be genetically dissected in quantitative resistance loci (QRL). To unravel the molecular basis of QRL in potato (Solanum tuberosum), we employed the model plant Arabidopsis thaliana for functional analysis of natural variants of potato allene oxide synthase 2 (StAOS2). StAOS2 is a candidate gene for QRL on potato chromosome XI against the oömycete Phytophthora infestans causing late blight, and the bacterium Erwinia carotovora ssp. atroseptica causing stem black leg and tuber soft rot, both devastating diseases in potato cultivation. StAOS2 encodes a cytochrome P450 enzyme that is essential for biosynthesis of the defense signaling molecule jasmonic acid. Allele non-specific dsRNAi-mediated silencing of StAOS2 in potato drastically reduced jasmonic acid production and compromised quantitative late blight resistance. Five natural StAOS2 alleles were expressed in the null Arabidopsis aos mutant under control of the Arabidopsis AOS promoter and tested for differential complementation phenotypes. The aos mutant phenotypes evaluated were lack of jasmonates, male sterility and susceptibility to Erwinia carotovora ssp. carotovora. StAOS2 alleles that were associated with increased disease resistance in potato complemented all aos mutant phenotypes better than StAOS2 alleles associated with increased susceptibility. First structure models of ‘quantitative resistant’ versus ‘quantitative susceptible’ StAOS2 alleles suggested potential mechanisms for their differential activity. Our results demonstrate how a candidate gene approach in combination with using the homologous Arabidopsis mutant as functional reporter can help to dissect the molecular basis of complex traits in non model crop plants.
Electronic supplementary material
The online version of this article (doi:10.1007/s00425-008-0737-x) contains supplementary material, which is available to authorized users.
PMCID: PMC2440949  PMID: 18431595
Black leg; Jasmonic acid; Late blight; Natural variation; Potato; Quantitative resistance; Soft rot
11.  Exon skipping induced by cold stress in a potato invertase gene transcript. 
Nucleic Acids Research  1996;24(12):2347-2351.
We show that two invertase genes in potato, like most other plant invertase genes, include a very short second exon of 9 bp which encodes the central three amino acids of a motif highly conserved in invertases of diverse origin. This mini-exon is one of the smallest known in plants and pre-mRNA from these genes may be susceptible to alternative splicing, because of a potential requirement for specialized interaction with the splicing machinery to ensure correct processing for the production of a mature mRNA. No evidence of aberrant post-transcriptional processing was observed during normal invertase gene expression in potato. The fidelity of post-transcriptional processing of the pre-mRNA from one of the genes was perturbed by cold stress, resulting in the deletion of the mini-exon from some transcripts. This alternative splicing event occurred under cold stress in both leaf and stem, but was not induced by wounding. This adds an example of exon skipping and the induction of alternative processing by cold stress to the small number of transcripts which have been shown to exhibit alternative splicing in plants. The differential sensitivity of post-transcriptional processing to cold stress observed for the two transcripts examined will permit further dissection of the nucleotide sequence requirements for their accurate splicing.
PMCID: PMC145944  PMID: 8710506
12.  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.
PMCID: PMC3550536  PMID: 23573000
Cold storage; Low temperature sweetening; Potato; Pyruvate decarboxylase; Transgenic
13.  Statistical epistasis between candidate gene alleles for complex tuber traits in an association mapping population of tetraploid potato 
Association mapping using DNA-based markers is a novel tool in plant genetics for the analysis of complex traits. Potato tuber yield, starch content, starch yield and chip color are complex traits of agronomic relevance, for which carbohydrate metabolism plays an important role. At the functional level, the genes and biochemical pathways involved in carbohydrate metabolism are among the best studied in plants. Quantitative traits such as tuber starch and sugar content are therefore models for association genetics in potato based on candidate genes. In an association mapping experiment conducted with a population of 243 tetraploid potato varieties and breeding clones, we previously identified associations between individual candidate gene alleles and tuber starch content, starch yield and chip quality. In the present paper, we tested 190 DNA markers at 36 loci scored in the same association mapping population for pairwise statistical epistatic interactions. Fifty marker pairs were associated mainly with tuber starch content and/or starch yield, at a cut-off value of q ≤ 0.20 for the experiment-wide false discovery rate (FDR). Thirteen marker pairs had an FDR of q ≤ 0.10. Alleles at loci encoding ribulose-bisphosphate carboxylase/oxygenase activase (Rca), sucrose phosphate synthase (Sps) and vacuolar invertase (Pain1) were most frequently involved in statistical epistatic interactions. The largest effect on tuber starch content and starch yield was observed for the paired alleles Pain1-8c and Rca-1a, explaining 9 and 10% of the total variance, respectively. The combination of these two alleles increased the means of tuber starch content and starch yield. Biological models to explain the observed statistical epistatic interactions are discussed.
Electronic supplementary material
The online version of this article (doi:10.1007/s00122-010-1389-3) contains supplementary material, which is available to authorized users.
PMCID: PMC2955219  PMID: 20603706
14.  Association genetics in Solanum tuberosum provides new insights into potato tuber bruising and enzymatic tissue discoloration 
BMC Genomics  2011;12:7.
Most agronomic plant traits result from complex molecular networks involving multiple genes and from environmental factors. One such trait is the enzymatic discoloration of fruit and tuber tissues initiated by mechanical impact (bruising). Tuber susceptibility to bruising is a complex trait of the cultivated potato (Solanum tuberosum) that is crucial for crop quality. As phenotypic evaluation of bruising is cumbersome, the application of diagnostic molecular markers would empower the selection of low bruising potato varieties. The genetic factors and molecular networks underlying enzymatic tissue discoloration are sparsely known. Hitherto there is no association study dealing with tuber bruising and diagnostic markers for enzymatic discoloration are rare.
The natural genetic diversity for bruising susceptibility was evaluated in elite middle European potato germplasm in order to elucidate its molecular basis. Association genetics using a candidate gene approach identified allelic variants in genes that function in tuber bruising and enzymatic browning. Two hundred and five tetraploid potato varieties and breeding clones related by descent were evaluated for two years in six environments for tuber bruising susceptibility, specific gravity, yield, shape and plant maturity. Correlations were found between different traits. In total 362 polymorphic DNA fragments, derived from 33 candidate genes and 29 SSR loci, were scored in the population and tested for association with the traits using a mixed model approach, which takes into account population structure and kinship. Twenty one highly significant (p < 0.001) and robust marker-trait associations were identified.
The observed trait correlations and associated marker fragments provide new insight in the molecular basis of bruising susceptibility and its natural variation. The markers diagnostic for increased or decreased bruising susceptibility will facilitate the combination of superior alleles in breeding programs. In addition, this study presents novel candidates that might control enzymatic tissue discoloration and tuber bruising. Their validation and characterization will increase the knowledge about the underlying biological processes.
PMCID: PMC3023753  PMID: 21208436
15.  Expression profiling of potato germplasm differentiated in quality traits leads to the identification of candidate flavour and texture genes 
Journal of Experimental Botany  2008;59(15):4219-4231.
Quality traits such as flavour and texture are assuming a greater importance in crop breeding programmes. This study takes advantage of potato germplasm differentiated in tuber flavour and texture traits. A recently developed 44 000-element potato microarray was used to identify tuber gene expression profiles that correspond to differences in tuber flavour and texture as well as carotenoid content and dormancy characteristics. Gene expression was compared in two Solanum tuberosum group Phureja cultivars and two S. tuberosum group Tuberosum cultivars; 309 genes were significantly and consistently up-regulated in Phureja, whereas 555 genes were down-regulated. Approximately 46% of the genes in these lists can be identified from their annotation and amongst these are candidates that may underpin the Phureja/Tuberosum trait differences. For example, a clear difference in the cooked tuber volatile profile is the higher level of the sesquiterpene α-copaene in Phureja compared with Tuberosum. A sesquiterpene synthase gene was identified as being more highly expressed in Phureja tubers and its corresponding full-length cDNA was demonstrated to encode α-copaene synthase. Other potential ‘flavour genes’, identified from their differential expression profiles, include those encoding branched-chain amino acid aminotransferase and a ribonuclease suggesting a mechanism for 5′-ribonucleotide formation in potato tubers on cooking. Major differences in the expression levels of genes involved in cell wall biosynthesis (and potentially texture) were also identified, including genes encoding pectin acetylesterase, xyloglucan endotransglycosylase and pectin methylesterase. Other gene expression differences that may impact tuber carotenoid content and tuber life-cycle phenotypes are discussed.
PMCID: PMC2639024  PMID: 18987392
α-Copaene; flavour; microarray; Solanum tuberosum group Phureja; Solanum tuberosum group Tuberosum; texture; volatile
16.  Structure and expression of GSL1 and GSL2 genes encoding gibberellin stimulated-like proteins in diploid and highly heterozygous tetraploid potato reveals their highly conserved and essential status 
BMC Genomics  2014;15:2.
GSL1 and GSL2, Gibberellin Stimulated-Like proteins (also known as Snakin-1 and Snakin-2), are cysteine-rich peptides from potato (Solanum tuberosum L.) with antimicrobial properties. Similar peptides in other species have been implicated in diverse biological processes and are hypothesised to play a role in several aspects of plant development, plant responses to biotic or abiotic stress through their participation in hormone crosstalk, and redox homeostasis. To help resolve the biological roles of GSL1 and GSL2 peptides we have undertaken an in depth analysis of the structure and expression of these genes in potato.
We have characterised the full length genes for both GSL1 (chromosome 4) and GSL2 (chromosome 1) from diploid and tetraploid potato using the reference genome sequence of potato, coupled with further next generation sequencing of four highly heterozygous tetraploid cultivars. The frequency of SNPs in GSL1 and GSL2 were very low with only one SNP every 67 and 53 nucleotides in exon regions of GSL1 and GSL2, respectively. Analysis of comprehensive RNA-seq data substantiated the role of specific promoter motifs in transcriptional control of gene expression. Expression analysis based on the frequency of next generation sequence reads established that GSL2 was expressed at a higher level than GSL1 in 30 out of 32 tissue and treatment libraries. Furthermore, both the GSL1 and GSL2 genes exhibited constitutive expression that was not up regulated in response to biotic or abiotic stresses, hormone treatments or wounding. Potato transformation with antisense knock-down expression cassettes failed to recover viable plants.
The potato GSL1 and GSL2 genes are very highly conserved suggesting they contribute to an important biological function. The known antimicrobial activity of the GSL proteins, coupled with the FPKM analysis from RNA-seq data, implies that both genes contribute to the constitutive defence barriers in potatoes. The lethality of antisense knock-down expression of GSL1 and GSL2, coupled with the rare incidence of SNPs in these genes, suggests an essential role for this gene family. These features are consistent with the GSL protein family playing a role in several aspects of plant development in addition to plant defence against biotic stresses.
PMCID: PMC3890649  PMID: 24382166
GSL1; GSL2; Gibberellin stimulated-like proteins; Potato; Snakin; Genome analysis; Transcriptional expression
17.  From QTL to candidate gene: Genetical genomics of simple and complex traits in potato using a pooling strategy 
BMC Genomics  2010;11:158.
Utilization of the natural genetic variation in traditional breeding programs remains a major challenge in crop plants. The identification of candidate genes underlying, or associated with, phenotypic trait QTLs is desired for effective marker assisted breeding. With the advent of high throughput -omics technologies, screening of entire populations for association of gene expression with targeted traits is becoming feasible but remains costly. Here we present the identification of novel candidate genes for different potato tuber quality traits by employing a pooling approach reducing the number of hybridizations needed. Extreme genotypes for a quantitative trait are collected and the RNA from contrasting bulks is then profiled with the aim of finding differentially expressed genes.
We have successfully implemented the pooling strategy for potato quality traits and identified candidate genes associated with potato tuber flesh color and tuber cooking type. Elevated expression level of a dominant allele of the β-carotene hydroxylase (bch) gene was associated with yellow flesh color through mapping of the gene under a major QTL for flesh color on chromosome 3. For a second trait, a candidate gene with homology to a tyrosine-lysine rich protein (TLRP) was identified based on allele specificity of the probe on the microarray. TLRP was mapped on chromosome 9 in close proximity to a QTL for potato cooking type strengthening its significance as a candidate gene. Furthermore, we have performed a profiling experiment targeting a polygenic trait, by pooling individual genotypes based both on phenotypic and marker data, allowing the identification of candidate genes associated with the two different linkage groups.
A pooling approach for RNA-profiling with the aim of identifying novel candidate genes associated with tuber quality traits was successfully implemented. The identified candidate genes for tuber flesh color (bch) and cooking type (tlrp) can provide useful markers for breeding schemes in the future. Strengths and limitations of the approach are discussed.
PMCID: PMC2843620  PMID: 20210995
18.  Effects of Nitrogen and Sulfur Fertilization on Free Amino Acids, Sugars, and Acrylamide-Forming Potential in Potato 
Nitrogen (N) fertilizer is used routinely in potato (Solanum tuberosum) cultivation to maximize yield. However, it also affects sugar and free amino acid concentrations in potato tubers, and this has potential implications for food quality and safety because free amino acids and reducing sugars participate in the Maillard reaction during high-temperature cooking and processing. This results in the formation of color, aroma, and flavor compounds, but also some undesirable contaminants, including acrylamide, which forms when the amino acid that participates in the final stages of the reaction is asparagine. Another mineral, sulfur (S), also has profound effects on tuber composition. In this study, 13 varieties of potato were grown in a field trial in 2010 and treated with different combinations of N and S. Potatoes were analyzed immediately after harvest to show the effect of N and S fertilization on concentrations of free asparagine, other free amino acids, sugars, and acrylamide-forming potential. The study showed that N application can affect acrylamide-forming potential in potatoes but that the effect is type- (French fry, chipping, and boiling) and variety-dependent, with most varieties showing an increase in acrylamide formation in response to increased N but two showing a decrease. S application reduced glucose concentrations and mitigated the effect of high N application on the acrylamide-forming potential of some of the French fry-type potatoes.
PMCID: PMC3711126  PMID: 23768004
acrylamide; asparagine; free amino acids; potato; Solanum tuberosum; sugars
19.  The ancestral activation promiscuity of ADP-glucose pyrophosphorylases from oxygenic photosynthetic organisms 
ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in the synthesis of glycogen in bacteria and starch in algae and plants. In oxygenic photosynthetic organisms, ADP-Glc PPase is mainly activated by 3-phosphoglycerate (3-PGA) and to a lesser extent by other metabolites. In this work, we analyzed the activation promiscuity of ADP-Glc PPase subunits from the cyanobacterium Anabaena PCC 7120, the green alga Ostreococcus tauri, and potato (Solanum tuberosum) tuber by comparing a specificity constant for 3-PGA, fructose-1,6-bisphosphate (FBP), fructose-6-phosphate, and glucose-6-phosphate.
The 3-PGA specificity constant for the enzymes from Anabaena (homotetramer), O. tauri, and potato tuber was considerably higher than for other activators. O. tauri and potato tuber enzymes were heterotetramers comprising homologous small and large subunits. Conversely, the O. tauri small subunit (OtaS) homotetramer was more promiscuous because its FBP specificity constant was similar to that for 3-PGA. To explore the role of both OtaS and OtaL (O. tauri large subunit) in determining the specificity of the heterotetramer, we knocked out the catalytic activity of each subunit individually by site-directed mutagenesis. Interestingly, the mutants OtaSD148A/OtaL and OtaS/OtaLD171A had higher specificity constants for 3-PGA than for FBP.
After gene duplication, OtaS seemed to have lost specificity for 3-PGA compared to FBP. This was physiologically and evolutionarily feasible because co-expression of both subunits restored the specificity for 3-PGA of the resulting heterotetrameric wild type enzyme. This widespread promiscuity seems to be ancestral and intrinsic to the enzyme family. Its presence could constitute an efficient evolutionary mechanism to accommodate the ADP-Glc PPase regulation to different metabolic needs.
PMCID: PMC3585822  PMID: 23433303
20.  Molecular and Physiological Properties Associated with Zebra Complex Disease in Potatoes and Its Relation with Candidatus Liberibacter Contents in Psyllid Vectors 
PLoS ONE  2012;7(5):e37345.
Zebra complex (ZC) disease on potatoes is associated with Candidatus Liberibacter solanacearum (CLs), an α-proteobacterium that resides in the plant phloem and is transmitted by the potato psyllid Bactericera cockerelli (Šulc). The name ZC originates from the brown striping in fried chips of infected tubers, but the whole plants also exhibit a variety of morphological features and symptoms for which the physiological or molecular basis are not understood. We determined that compared to healthy plants, stems of ZC-plants accumulate starch and more than three-fold total protein, including gene expression regulatory factors (e.g. cyclophilin) and tuber storage proteins (e.g., patatins), indicating that ZC-affected stems are reprogrammed to exhibit tuber-like physiological properties. Furthermore, the total phenolic content in ZC potato stems was elevated two-fold, and amounts of polyphenol oxidase enzyme were also high, both serving to explain the ZC-hallmark rapid brown discoloration of air-exposed damaged tissue. Newly developed quantitative and/or conventional PCR demonstrated that the percentage of psyllids in laboratory colonies containing detectable levels of CLs and its titer could fluctuate over time with effects on colony prolificacy, but presumed reproduction-associated primary endosymbiont levels remained stable. Potato plants exposed in the laboratory to psyllid populations with relatively low-CLs content survived while exposure of plants to high-CLs psyllids rapidly culminated in a lethal collapse. In conclusion, we identified plant physiological biomarkers associated with the presence of ZC and/or CLs in the vegetative potato plant tissue and determined that the titer of CLs in the psyllid population directly affects the rate of disease development in plants.
PMCID: PMC3355140  PMID: 22615987
21.  Glycosyltransferase efficiently controls phenylpropanoid pathway 
BMC Biotechnology  2008;8:25.
In a previous study, anthocyanin levels in potato plants were increased by manipulating genes connected with the flavonoid biosynthesis pathway. However, starch content and tuber yield were dramatically reduced in the transgenic plants, which over-expressed dihydroflavonol reductase (DFR).
Transgenic plants over-expressing dihydroflavonol reductase (DFR) were subsequently transformed with the cDNA coding for the glycosyltransferase (UGT) of Solanum sogarandinum in order to obtain plants with a high anthocyanin content without reducing tuber yield and quality. Based on enzyme studies, the recombinant UGT is a 7-O-glycosyltransferase whose natural substrates include both anthocyanidins and flavonols such as kaempferol and quercetin. In the super-transformed plants, tuber production was much higher than in the original transgenic plants bearing only the transgene coding for DFR, and was almost the same as in the control plants. The anthocyanin level was lower than in the initial plants, but still higher than in the control plants. Unexpectedly, the super-transformed plants also produced large amounts of kaempferol, chlorogenic acid, isochlorogenic acid, sinapic acid and proanthocyanins.
In plants over-expressing both the transgene for DFR and the transgene for UGT, the synthesis of phenolic acids was diverted away from the anthocyanin branch. This represents a novel approach to manipulating phenolic acids synthesis in plants.
PMCID: PMC2294120  PMID: 18321380
22.  Effects of In-furrow and Water-run Oxamyl on Paratrichodorus allius and Corky Ringspot Disease of Potato in the Klamath Basin 
Journal of Nematology  2010;42(1):1-7.
Corky ringspot disease (CRS) of potato (Solanum tuberosum) is caused by the tobacco rattle virus (TRV), which is vectored by stubby-root nematodes, Paratrichodorus spp. and Trichodorus spp., and is a significant threat to potato quality and production in many areas of the western United States. Between 2002 and 2005, fields with a history of CRS were planted to potato and treated with various combinations of in-furrow (IF) and chemigated (water run, WR) oxamyl [Methyl N'N'-dimethyl-N-[(methyl carbamoyl)oxy]-1-thiooxamimidate] applications. Soil samples were collected to determine how Paratrichodorus allius populations responded to the various treatment regimes (2002-2004); potato tubers were evaluated for symptoms of CRS in 2004-2005. Applications of oxamyl to potato (1.1 kg a.i./ha) did not cause significant mortality of P. allius but did prevent the populations from increasing. Oxamyl applications that began at 55 days after planting (DAP) or later did not control CRS and were not different from the untreated control. However, application schedules that began early-season, either IF at planting, early WR (33 – 41 DAP), or both, significantly reduced CRS expression in cv. Yukon Gold. Therefore, oxamyl applications must be made early in the growing season to be effective in controlling CRS. Effects of oxamyl on CRS may be due to nematostatic action that suppresses feeding activity during early field season when most virus transmission probably occurs.
PMCID: PMC3380511  PMID: 22736830
corky ringspot; CRS; oxamyl; population dynamics; potato; Solanum tuberosum; stubby-root nematodes; Paratrichodorus spp.; Trichodorus spp.; tobacco rattle virus; TRV; Yukon Gold
23.  Analysis of 90 Mb of the potato genome reveals conservation of gene structures and order with tomato but divergence in repetitive sequence composition 
BMC Genomics  2008;9:286.
The Solanaceae family contains a number of important crop species including potato (Solanum tuberosum) which is grown for its underground storage organ known as a tuber. Albeit the 4th most important food crop in the world, other than a collection of ~220,000 Expressed Sequence Tags, limited genomic sequence information is currently available for potato and advances in potato yield and nutrition content would be greatly assisted through access to a complete genome sequence. While morphologically diverse, Solanaceae species such as potato, tomato, pepper, and eggplant share not only genes but also gene order thereby permitting highly informative comparative genomic analyses.
In this study, we report on analysis 89.9 Mb of potato genomic sequence representing 10.2% of the genome generated through end sequencing of a potato bacterial artificial chromosome (BAC) clone library (87 Mb) and sequencing of 22 potato BAC clones (2.9 Mb). The GC content of potato is very similar to Solanum lycopersicon (tomato) and other dicotyledonous species yet distinct from the monocotyledonous grass species, Oryza sativa. Parallel analyses of repetitive sequences in potato and tomato revealed substantial differences in their abundance, 34.2% in potato versus 46.3% in tomato, which is consistent with the increased genome size per haploid genome of these two Solanum species. Specific classes and types of repetitive sequences were also differentially represented between these two species including a telomeric-related repetitive sequence, ribosomal DNA, and a number of unclassified repetitive sequences. Comparative analyses between tomato and potato at the gene level revealed a high level of conservation of gene content, genic feature, and gene order although discordances in synteny were observed.
Genomic level analyses of potato and tomato confirm that gene sequence and gene order are conserved between these solanaceous species and that this conservation can be leveraged in genomic applications including cross-species annotation and genome sequencing initiatives. While tomato and potato share genic features, they differ in their repetitive sequence content and composition suggesting that repetitive sequences may have a more significant role in shaping speciation than previously reported.
PMCID: PMC2442093  PMID: 18554403
24.  SolRgene: an online database to explore disease resistance genes in tuber-bearing Solanum species 
BMC Plant Biology  2011;11:116.
The cultivated potato (Solanum tuberosum L.) is an important food crop, but highly susceptible to many pathogens. The major threat to potato production is the Irish famine pathogen Phytophthora infestans, which causes the devastating late blight disease. Potato breeding makes use of germplasm from wild relatives (wild germplasm) to introduce resistances into cultivated potato. The Solanum section Petota comprises tuber-bearing species that are potential donors of new disease resistance genes. The aim of this study was to explore Solanum section Petota for resistance genes and generate a widely accessible resource that is useful for studying and implementing disease resistance in potato.
The SolRgene database contains data on resistance to P. infestans and presence of R genes and R gene homologues in Solanum section Petota. We have explored Solanum section Petota for resistance to late blight in high throughput disease tests under various laboratory conditions and in field trials. From resistant wild germplasm, segregating populations were generated and assessed for the presence of resistance genes. All these data have been entered into the SolRgene database. To facilitate genetic and resistance gene evolution studies, phylogenetic data of the entire SolRgene collection are included, as well as a tool for generating phylogenetic trees of selected groups of germplasm. Data from resistance gene allele-mining studies are incorporated, which enables detection of R gene homologs in related germplasm. Using these resources, various resistance genes have been detected and some of these have been cloned, whereas others are in the cloning pipeline. All this information is stored in the online SolRgene database, which allows users to query resistance data, sequences, passport data of the accessions, and phylogenic classifications.
Solanum section Petota forms the basis of the SolRgene database, which contains a collection of resistance data of an unprecedented size and precision. Complemented with R gene sequence data and phylogenetic tools, SolRgene can be considered the primary resource for information on R genes from potato and wild tuber-bearing relatives.
PMCID: PMC3166922  PMID: 21851635
25.  Normal Operating Range of Bacterial Communities in Soil Used for Potato Cropping 
In this study, the impacts of six potato (Solanum tuberosum) cultivars with different tuber starch allocations (including one genetically modified [GM] line) on the bacterial communities in field soil were investigated across two growth seasons interspersed with 1 year of barley cultivation, using quantitative PCR, clone library, and PCR-denaturing gradient gel electrophoresis (DGGE) analyses. It was hypothesized that the modifications in the tuber starch contents of these plants, yielding changed root growth rates and exudation patterns, might have elicited altered bacterial communities in the soil. The data showed that bacterial abundances in the bulk soil varied over about 2 orders of magnitude across the 3 years. As expected, across all cultivars, positive potato rhizosphere effects on bacterial abundances were noted in the two potato years. The bulk soil bacterial community structures revealed progressive shifts across time, and moving-window analysis revealed a 60% change over the total experiment. Consistent with previous findings, the community structures in the potato rhizosphere compartments were mainly affected by the growth stage of the plants and, to a lesser extent, by plant cultivar type. The data from the soil under the non-GM potato lines were then taken to define the normal operating range (NOR) of the microbiota under potatoes. Interestingly, the bacterial communities under the GM potato line remained within this NOR. In regard to the bacterial community compositions, particular bacterial species in the soil appeared to be specific to (i) the plant species under investigation (barley versus potato) or, with respect to potatoes, (ii) the plant growth stage. Members of the genera Arthrobacter, Streptomyces, Rhodanobacter, and Dokdonella were consistently found only at the flowering potato plants in both seasons, whereas Rhodoplanes and Sporosarcina were observed only in the soil planted to barley.
PMCID: PMC3568629  PMID: 23220956

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