Two Cytochrome P450 Monooxygenases Catalyze Early Hydroxylation Steps in the Potato Steroid Glycoalkaloid Biosynthetic Pathway

α-Solanine and α-chaconine, steroidal glycoalkaloids (SGAs) found in potato (Solanum tuberosum), are among the best-known secondary metabolites in food crops. At low concentrations in potato tubers, SGAs are distasteful; however, at high concentrations, SGAs are harmful to humans and animals. Here, we show that POTATO GLYCOALKALOID BIOSYNTHESIS1 (PGA1) and PGA2, two genes that encode cytochrome P450 monooxygenases (CYP72A208 and CYP72A188), are involved in the SGA biosynthetic pathway, respectively. The knockdown plants of either PGA1 or PGA2 contained very little SGA, yet vegetative growth and tuber production were not affected. Analyzing metabolites that accumulated in the plants and produced by in vitro enzyme assays revealed that PGA1 and PGA2 catalyzed the 26- and 22-hydroxylation steps, respectively, in the SGA biosynthetic pathway. The PGA-knockdown plants had two unique phenotypic characteristics: The plants were sterile and tubers of these knockdown plants did not sprout during storage. Functional analyses of PGA1 and PGA2 have provided clues for controlling both potato glycoalkaloid biosynthesis and tuber sprouting, two traits that can significantly impact potato breeding and the industry.Potato (Solanum tuberosum) is the world’s fourth most important food crop after maize (Zea mays), rice (Oryza sativa), and wheat (Triticum aestivum). Potato steroidal glycoalkaloids (SGAs) are abundant poisons in tuber sprouts and green tubers and are described as bitter tasting, burning, scratchy, or acrid (Friedman, 2006; Ginzberg et al., 2009; Taylor et al., 2007). SGAs in the tuber are induced by exposure to light, low temperature, and mechanical injury (Valkonen et al., 1996). Producers and consumers have called for the removal of SGAs from potatoes. Only potato with SGAs of major food crops has such broad industry consensus on the need to solve this important worldwide problem. Controlling the SGA content is also important for potato breeding. Wild germplasm has been used in potato breeding programs as a source of pest and disease resistance. Since high SGA concentrations are found in most wild tuber-bearing species, introgression of wild germplasm may increase the risk for high SGA levels. Although the initial concentrations are still low in new breeding lines, dangerous levels of SGAs can accumulate due to environmental factors, pathogen infections, and postharvest treatments (Valkonen et al., 1996).SGAs are biosynthesized from a common precursor, cholesterol (CHR; Sawai et al., 2014), but little is known about intermediates and enzymes in the SGA biosynthetic pathway. To change a biosynthetic flow to CHR and decrease SGA contents, transgenic potatoes overexpressing a heterologous soybean sterol methyltransferase gene were produced (Arnqvist et al., 2003). Three genes responsible for glycosylating potato SGA have been identified (McCue et al., 2005, 2006, 2007). However, changing the expression of the sterol methyltransferase or glycosyltransferase genes does not effectively decrease SGA levels. To control the SGA content of potato, we focused on the biosynthetic steps from CHR to the aglycone, solanidine. Few details about the biosynthetic pathway are verified; however, the pathway is hypothesized to require at least three oxidization steps at positions C16, C22, and C26 of CHR structure and the addition of one nitrogen atom at the position C26 (Fig. 1; Kaneko et al., 1977; Erich, 1983; Eich, 2008). The later step was shown to be another oxidation and an amination reaction at the position C26 (Ohyama et al., 2013). Here, we identified two cytochrome P450 monooxygenase (CYPs) genes, POTATO GLYCOALKALOID BIOSYNTHESIS1 (PGA1) and PGA2 that encode enzymes mediating two oxidation steps. Silencing PGA1 and PGA2 resulted in a significant reduction in SGA composition and the creation of novel phenotypes, including the suppression of flower development and tuber sprouting. Sprouting reduces the quality and yield of potato tubers in storage. Suppression of tuber sprouting is of significant benefit to the industry for the long-term storage of tubers. Thus, controlling tuber sprouting is another important objective in potato breeding (Sonnewald and Sonnewald, 2014).Open in a separate windowFigure 1.Biosynthetic pathway for SGAs in potato. The structures of CHR and solanidine, two biosynthetic intermediates of potato SGAs, are shown with the structures of the SGA products. Circles indicate putative carbon positions that are oxidized in the hypothesized pathway.     

Potato Glycoalkaloids: A Burden or a Blessing?

Steroidal glycoalkaloids (SGAs) are produced following the general steroid biosynthesis pathway, starting from acetyl-coenzyme A and followed by the intermediates mevalonic acid, squalene, cycloartenol, and cholesterol. α-Chaconine and α-solanine are the main SGAs of the cultivated potato (Solanum tuberosum), whereas many other SGAs are known in the wild potato species. Low concentrations of SGAs improve the taste of potato, but concentrations greater than 200 mg/kg can have toxic effects on animals and humans. SGAs have antimicrobial activity and confer resistance to some insects, but many such pests of potato are not greatly affected. Certain environmental conditions and wounding enhance SGA accumulation in tubers in the field and storage. Low production of SGAs is a dominant character inherited in a relatively simple manner and can be selected for in potato-breeding programs, whereas the use of wild potato germplasm tends to increase the SGA accumulation in the breeding lines. Further efforts are likely to be directed toward the reduction of the SGA content in the edible potato products through breeding and biotechnological methodologies, whereas potato genotypes with high SGA production may be developed for use in the pharmaceutical industry.     

Allelic variation in genes contributing to glycoalkaloid biosynthesis in a diploid interspecific population of potato

Key message

Variation for allelic state within genes of both primary and secondary metabolism influences the quantity and quality of steroidal glycoalkaloids produced in potato leaves.

Abstract

Genetic factors associated with the biosynthesis and accumulation of steroidal glycoalkaloids (SGAs) in potato were addressed by a candidate gene approach and whole genome single nucleotide polymorphism (SNP) genotyping. Allelic sequences spanning coding regions of four candidate genes [3-hydroxy-3-methylglutaryl coenzyme A reductase 2 (HMG2); 2,3-squalene epoxidase; solanidine galactosyltransferase; and solanidine glucosyltransferase (SGT2)] were obtained from two potato species differing in SGA composition: Solanum chacoense (chc 80-1) and Solanum tuberosum group Phureja (phu DH). An F₂ population was genotyped and foliar SGAs quantified. The concentrations of α-solanine, α-chaconine, leptine I, leptine II and total SGAs varied broadly among F₂ individuals. F₂ plants with chc 80-1 alleles for HMG2 or SGT2 accumulated significantly greater leptines and total SGAs compared to plants with phu DH alleles. Plants with chc 80-1 alleles at both loci expressed the greatest levels of total SGAs, α-solanine and α-chaconine. A significant positive correlation was found between α-solanine and α-chaconine accumulation as well as between leptine I and leptine II. A whole genome SNP genotyping analysis of an F₂ subsample verified the importance of chc 80-1 alleles at HMG2 and SGT2 for SGA synthesis and accumulation and suggested additional candidate genes including some previously associated with SGA production. Loci on five and seven potato pseudochromosomes were associated with synthesis and accumulation of SGAs, respectively. Two loci, on pseudochromosomes 1 and 6, explained phenotypic segregation of α-solanine and α-chaconine synthesis. Knowledge of the genetic factors influencing SGA production in potato may assist breeding for pest resistance.     

Biosynthesis of steroidal alkaloids in Solanaceae plants: Incorporation of 3β-hydroxycholest-5-en-26-al into tomatine with tomato seedlings

The C-26 amino group of tomatine, a representative Solanaceae steroidal alkaloid, is introduced in an early step of its biosynthesis from cholesterol. We recently proposed a transamination mechanism for the C-26 amination as opposed to the previously proposed mechanism involving a nitrogen nucleophilic displacement. In the present study, a deuterium labeled C-26 aldehyde, (24,24,27,27,27-²H₅)-3β-hydroxycholest-5-en-26-al, was synthesized and fed to a tomato (Solanum lycopersicum) seedling. LC–MS analysis of the biosynthesized tomatine indicated that the labeled aldehyde was incorporated into tomatine. The finding strongly supports the intermediacy of the aldehyde and the transamination mechanism during C-26 amination.     

GLYCOALKALOID METABOLISM1 is required for steroidal alkaloid glycosylation and prevention of phytotoxicity in tomato

Steroidal alkaloids (SAs) are triterpene-derived specialized metabolites found in members of the Solanaceae family that provide plants with a chemical barrier against a broad range of pathogens. Their biosynthesis involves the action of glycosyltransferases to form steroidal glycoalkaloids (SGAs). To elucidate the metabolism of SGAs in the Solanaceae family, we examined the tomato (Solanum lycopersicum) GLYCOALKALOID METABOLISM1 (GAME1) gene. Our findings imply that GAME1 is a galactosyltransferase, largely performing glycosylation of the aglycone tomatidine, resulting in SGA production in green tissues. Downregulation of GAME1 resulted in an almost 50% reduction in α-tomatine levels (the major SGA in tomato) and a large increase in its precursors (i.e., tomatidenol and tomatidine). Surprisingly, GAME1-silenced plants displayed growth retardation and severe morphological phenotypes that we suggest occur as a result of altered membrane sterol levels caused by the accumulation of the aglycone tomatidine. Together, these findings highlight the role of GAME1 in the glycosylation of SAs and in reducing the toxicity of SA metabolites to the plant cell.     

Metabolomics-assisted refinement of the pathways of steroidal glycoalkaloid biosynthesis in the tomato clade

Steroidal glycoalkaloids (SGAs) are nitrogen-con-taining secondary metabolites of the Solanum species, which are known to have large chemical and bioactive diversity in nature. While recent effort and ...     

Sterol Side Chain Reductase 2 Is a Key Enzyme in the Biosynthesis of Cholesterol,the Common Precursor of Toxic Steroidal Glycoalkaloids in Potato

Potatoes (Solanum tuberosum) contain α-solanine and α-chaconine, two well-known toxic steroidal glycoalkaloids (SGAs). Sprouts and green tubers accumulate especially high levels of SGAs. Although SGAs were proposed to be biosynthesized from cholesterol, the biosynthetic pathway for plant cholesterol is poorly understood. Here, we identify sterol side chain reductase 2 (SSR2) from potato as a key enzyme in the biosynthesis of cholesterol and related SGAs. Using in vitro enzyme activity assays, we determined that potato SSR2 (St SSR2) reduces desmosterol and cycloartenol to cholesterol and cycloartanol, respectively. These reduction steps are branch points in the biosynthetic pathways between C-24 alkylsterols and cholesterol in potato. Similar enzymatic results were also obtained from tomato SSR2. St SSR2-silenced potatoes or St SSR2-disrupted potato generated by targeted genome editing had significantly lower levels of cholesterol and SGAs without affecting plant growth. Our results suggest that St SSR2 is a promising target gene for breeding potatoes with low SGA levels.     

Optimization of Glycoalkaloid Analysis for Use in Industrial Potato Fruit Juice Downstreaming

Annually, within the European Union about 1.7 million tons of starch is produced by processing over 8 million tons of potato tubers, Solanum tuberosum. In recent years, the potato protein content has gained tremendous industrial interest, since these proteins have excellent nutritional value. As naturally occurring, secondary plant metabolites steroidal potato glycoalkaloids are formed in potatoes. The two major glycoalkaloids in potatoes are α‐solanine and α‐chaconine. Because of the significant toxicity of the glycoalkaloids for human and for animal nutrition it was essential to develop efficient extraction processes. The need for an easy, fast, sensitive and reliable glycoalkaloid assay at the very beginning of the production chain is obvious. In this study an efficient analytical assay for potato glycoalkaloids from powdery protein samples under industrially relevant conditions is described: sample extraction, analyte pre‐purification, and final HPLC analysis. An acetic acid extraction/homogenization process was used for glycoalkaloid extraction from potato protein samples. The extracts were purified by means of solid phase extraction cartridges using the different washing steps developed in this study. The final determination was performed through an HPLC method using a Reprosil‐Pur NH₂ column. The limit of detection was 5 μg/mL for α‐solanine and α‐chaconine, respectively, corresponding to concentrations of 20 ppm in potato protein powder.     

Evolution of nematode-resistant <Emphasis Type="Italic">Mi</Emphasis>-<Emphasis Type="Italic">1</Emphasis> gene homologs in three species of <Emphasis Type="Italic">Solanum</Emphasis>

Plants have evolved several defense mechanisms, including resistance genes. Resistance to the root-knot nematode Meloidogyne incognita has been found in wild plant species. The molecular basis for this resistance has been best studied in the wild tomato Solanum peruvianum and it is based on a single dominant gene, Mi-1.2, which is found in a cluster of seven genes. This nematode attacks fiercely several crops, including potatoes. The genomic arrangement, number of copies, function and evolution of Mi-1 homologs in potatoes remain unknown. In this study, we analyzed partial genome sequences of the cultivated potato species S. tuberosum and S. phureja and identified 59 Mi-1 homologs. Mi-1 homologs in S. tuberosum seem to be arranged in clusters and located on chromosome 6 of the potato genome. Previous studies have suggested that Mi-1 genes in tomato evolved rapidly by frequent sequence exchanges among gene copies within the same cluster, losing orthologous relationships. In contrast, Mi-1 homologs from cultivated potato species (S. tuberosum and S. phureja) seem to have evolved by a birth-and-death process, in which genes evolve mostly by mutations and interallelic recombinations in addition to sequence exchanges.     

Population dynamics of the potato tuber moth on processing tomatoes in Israel

The potato tuber moth (PTM),Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae), is a major pest of processing tomatoes,Lycopersicon esculentum Mill. (Solanaceae), in Israel. The larvae penetrate the tomato fruit through the stem end and present a serious threat to crop quality. Foliage and fruit samples were taken in nine commercial tomato fields located in Israel's three main tomato growing areas, two of which are potato growing areas as well. PTM was not found where potatoes were absent. Potato harvest in nearby fields was found to be the most significant factor affecting seasonal trends in PTM population density in tomatoes. All four larval instars were found in foliage on all sampling dates. Significantly higher proportions of first instars were found during the population density increase which followed potato harvest. Damaged fruits did not contain first instar larvae, indicating that PTM never undergoes complete development within tomato fruit. Fruit damage levels at harvest were positively correlated to the peak mean population densities on foliage and the date they were observed. In tomato fields not adjacent to potatoes, infestation was first observed at the edge of the field. Both before and after the potato harvest in nearby fields, population density at the edge of the field was significantly higher than at the center. In tomato fields adjacent to potatoes, no significant differences were found between population densities at the edge and center before the potatoes were harvested. After the potato harvest, population density at the center of tomato fields was higher than at the edge. Deceased, October 1988     

The primary in vivo steroidal alkaloid glucosyltransferase from potato

To provide tools for breeders to control the steroidal glycoalkaloid (SGA) pathway in potato, we have investigated the steroidal alkaloid glycosyltransferase (Sgt) gene family. The committed step in the SGA pathway is the glycosylation of solanidine by either UDP-glucose or UDP-galactose leading to α-chaconine or α-solanine, respectively. The Sgt2 gene was identified by deduced protein sequence homology to the previously identified Sgt1 gene. SGT1 has glucosyltransferase activity in vitro, but in vivo serves as the UDP-galactose:solanidine galactosyltransferase. Two alleles of the Sgt2 gene were isolated and its function was established with antisense transgenic lines and in vitro assays of recombinant protein. In tubers of transgenic potato (Solanum tuberosum) cvs. Lenape and Desirée expressing an antisense Sgt2 gene construct, accumulation of α-solanine was increased and α-chaconine was reduced. Studies with recombinant SGT2 protein purified from yeast show that SGT2 glycosylation activity is highly specific for UDP-glucose as a sugar donor. This data establishes the function of the gene product (SGT2), as the primary UDP-glucose:solanidine glucosyltransferase in vivo.     

Autonomous cell death, temperature sensitivity and the genetic control associated with resistance to cucumber mosaic virus (CMV) in diploid potatoes (Solanum spp.)

Cucumber mosaic virus (CMV) is a commonly occurring plant virus that causes severe damage in many crops, including the diploid crop species tomato and pepper (Lycopersicon spp. and Capsicum spp., respectively) of the family Solanaceae, but it is neither common nor economically important in cultivated potatoes (Solanum tuberosum; Solanaceae). Resistance to CMV was examined in two diploid (2n=2x=24), highly heterozygous potato populations (Solanum spp.; Solanaceae) consisting of 76 and 126 progeny. Resistance to long-distance transport of CMV controlled by one locus with a major effect and functional at a low temperature (18°C) but overcome at a high temperature (28°C) was identified in one population. In the other population, resistance was controlled by two loci with major effects. In both populations, additional genes with minor effects were probably also involved. Induced resistance to CMV, associated with autonomously developing cell death lesions (Anl) previously not known in potato, was expressed in one parental line. The mechanisms of resistance to CMV may be associated with an inherent or developmental lack of host factors required for compatible CMV-host interactions in viral long distance transport and/or inability of CMV to efficiently suppress the host gene silencing mechanism in potatoes. Polyploidy (gene dose) and high heterozygosity (multiple homologous genes) of potato cultivars may be significant in conferring the durable resistance to CMV. These data provide explanations why CMV is not common and economically important in cultivated potatoes, even though CMV commonly occurs in other crops, weeds and wild plants in potato production areas. Received: 11 February 1999 / Accepted: 25 March 1999     

Production of diosgenin from yellow ginger (<Emphasis Type="Italic">Dioscorea zingiberensis</Emphasis> C. H. Wright) saponins by commercial cellulase

A commercial cellulase was first assessed to be effective in hydrolyzing glycosyl at the C-3 and C-26 positions in steroidal saponins from yellow ginger (Dioscorea zingiberensis C. H. Wright) to diosgenin, a very important chemical in the pharmaceutical industry. The effect of different parameters on enzyme hydrolysis was further investigated by systematically varying them. The highest yield was achieved when the hydrolysis ran at 55°C and pH 5.0 with an enzyme to substrate ratio of 15 × 10³ U/g. The biotransformed products identified using TLC and HPLC confirmed that the cellulase was capable of releasing diosgenin from steroidal saponins. Moreover, the biotransformation process was explored by LC-MS and LC-MS/MS analysis. Enzymatic hydrolysis together with 40 % of the original sulphuric acid used increased the diosgenin yield by 15.4 ± 2.7% than traditional method. Therefore, the commercial cellulase may serve as a promising tool for industrial diosgenin production and for further use in saponin modification.     

Origin of chloroplast DNA diversity in the Andean potatoes

Summary Wide chloroplast DNA (ctDNA) diversity has been reported in the Andean cultivated tetraploid potato, Solanum tuberosum ssp. andigena. Andean diploid potatoes were analyzed in this study to elucidate the origin of the diverse ctDNA variation of the cultivated tetraploids. The ctDNA types of 58 cultivated diploid potatoes (S. stenotomum, S. goniocalyx and S. phureja), 35 accessions of S. sparsipilum, a diploid weed species, and 40 accessions of the wild or weed species, S. chacoense, were determined based on ctDNA restriction fragment patterns of BamHI, HindIII and PvuII. Several different ctDNA types were found in the cultivated potatoes as well as in weed and wild potato species; thus, intraspecific ctDNA variation may be common in both wild and cultivated potato species and perhaps in the higher plant kingdom as a whole. The ctDNA variation range of cultivated diploid potatoes was similar to that of the tetraploid potatoes, suggesting that the ctDNA diversity of the tetraploid potato could have been introduced from cultivated diploid potatoes. This provided further evidence that the Andean cultivated tetraploid potato, ssp. andigena, could have arisen many times from the cultivated diploid populations. The diverse but conserved ctDNA variation noted in the Andean potatoes may have occurred in the early stage of species differentiation of South American tuber-bearing Solanums.     

Conversion of Exogenous Cholesterol into Glycoalkaloids in Potato Shoots,Using Two Methods for Sterol Solubilisation

Steroidal glycoalkaloids (SGA) are toxic secondary metabolites naturally occurring in the potato, as well as in certain other Solanaceous plant species, such as tomato, eggplant and pepper. To investigate the steroidal origin of SGA biosynthesis, cut potato shoots were fed cholesterol labelled with deuterium (D) in the sterol ring structure (D₅- or D₆-labelled), or side chain (D₇-labelled), and analysed after three or five weeks. The labelled cholesterol and presence of D-labelled SGA were analysed by GC-MS and LC-MS/MS, respectively. When feeding D-labelled cholesterol solubilised in Tween-80, labelled cholesterol in free form became present in both leaves and stems, although the major part was recovered as steryl esters. Minor amounts of D-labelled SGA (α-solanine and α-chaconine) were identified in cholesterol-treated shoots, but not in blank controls, or in shoots fed D₆-27-hydroxycholesterol. Solubilising the labelled cholesterol in methyl-β-cyclodextrin instead of Tween-80 increased the levels of labelled SGA up to 100-fold, and about 1 mole% of the labelled cholesterol was recovered as labelled SGA in potato leaves. Both side chain and ring structure D labels were retained in SGA, showing that the entire cholesterol molecule is converted to SGA. However, feeding side chain D₇-labelled cholesterol resulted in D₅-labelled SGA, indicating that two hydrogen atoms were released during formation of the SGA nitrogen-containing ring system. Feeding with D₇-sitosterol did not produce any labelled SGA, indicating that cholesterol is a specific SGA precursor. In conclusion, we have demonstrated a superior performance of methyl-β-cyclodextrin for delivery of cholesterol in plant tissue feeding experiments, and given firm evidence for cholesterol as a specific sterol precursor of SGA in potato.     

Genomic structural differentiation in Solanum: comparative mapping of the A- and E-genomes

 A genetic map was constructed from an F₂ population of 76 individuals for the purpose of comparing the arrangement of loci in the A and E Solanum genomes. This progeny was derived from an interspecific cross between the species Solanum palustre×Solanum etuberosum, both of which are E-genome species. Two hundred and eighty one probes previously mapped in tomato and potato (A-genome, as postulated for diploid cultivated potato species by Matsubayashi 1991) disclosed 109 segregating loci in this population. Of these, 80 loci were linked in 19 linkage groups covering a total of 720.4 cM, with an average of 9 cM between markers. Although the genetic map of the E-genome showed conservation for most linkage groups with those of tomato and the A-genome, various translocations and possible inversions and transpositions were detected. It is evident that the accumulation of these structural changes in the E-genome is sufficient to cause the observed hybrid sterility. The major rearrangements in the E-genome included multiple translocations involving mosly linkage groups 2 and 8. Also a transposition was detected on group 9, with the same group-10 inversion distinguishing potato from tomato. Definitively groups 2, 8, 9 and 10, and possibly groups 1, 4 and 12, in the E-genome are structurally different from their homologues in the A-genome. In general, recombination values were larger in the E- than in the A-genome. The extensive structural differentiation of the E-genome with respect to that of potato and tomato justifies its present designation as a different genome, which is supported by previous chromosome-pairing studies. The difficult introgression of desirable traits from the Etuberosum species into potato can be explained by these structural differences. Received: 1 February 1998 / Accepted: 8 October 1998