Relating genes in the biosynthesis of the polyphenol composition of Andean colored potato collection

The objective of this study was to evaluate total antioxidant capacity (TAC), total phenolic content (TPH), and the identification of anthocyanidin and polyphenolic compounds in 13 colored potatoes collected from the Andean region of Bolivia, and understand how the chemical composition correlated with the botanical classification and molecular characterization of genes, ans (anthocyanidin synthase) and stan1 (Solanum tuberosum anthocyanidin synthase), associated with the synthesis of anthocyanidins. The results show the existence of a limited correlation between botanical classification, based on morphological identification and polyphenol composition. No association between genetic grouping of the ans and stan genes and botanical classification was found. However, it was possible to identify a correlation between the ans gene clades and the levels of anthocyanidins as well as of other polyphenols. Thus, this result confirms the concept that potato color can be used in the search for high polyphenol potato cultivars.


Introduction
Potatoes accumulate a great variety of secondary metabolites, including polyphenolic and many other phytochemical compounds, as a protection against the adverse effects of solar radiation, and injury of herbivorous insects or pathogens such as bacteria or fungi (Del Mar Verde M endez et al. 2004;Brown 2005). Polyphenolic compounds are able to form stable radicals and thereby act as radical scavengers and antioxidants. As antioxidants, polyphenols are expected to have potentially positive health benefits (Del Mar Verde M endez et al. 2004). Anthocyanidins is a group of polyphenols able to create red, purple, blue, or orange colors (Brown 2005).
Anthocyanidin biosynthesis is one of the most studied plant secondary metabolite pathways. There are many genes responsible for enzyme synthesis. For almost every biosynthetic step the corresponding enzymes have been isolated and a considerable body of knowledge is available on the mechanisms that regulate their expression in the plant cell (Deroles 2009). Two classes of genes are required for anthocyanidin biosynthesisthe structural genes, for example, ans and chi that code for the enzymes that are directly involved in the production of anthocyanidin and the regulatory genes, for example, stan1 and stan2, that control the transcription of structural genes (Al-Sane et al. 2011). The pathway for the anthocyanidin biosynthesis is shown in Figure 1.
This work is focused on comparing the chemical composition with botanical classification based on morphological characteristic and the similarities between the involved genes. Our hypothesis was that there is an association between the similarities, the involved genes, and the concentration patterns of anthocyanidins and antioxidants.

Collection of potatoes
Thirteen purple potatoes belonging to three species (Solanum curtilobum, S. ajanhuiri, and S. tuberosum subsp. andigenum) (Fig. 2) were harvested in April 2011 during one day at Quipaquipani Research and Training Center (PROINPA Foundation). The station is located 4 km south of the town of Viacha (coordinates 68°17′49″, 16°40′17″), Ingavi Province in the Department of La Paz 3870 m.a.s.l. Each sample was taken from separate clones grown at a section of a field. The sample was obtained through a random collection of potatoes from the growing section until 1 kg was obtained. The samples were stored in plastic bags at 5°C for 6 h and then transferred to the laboratory.

Extraction (TAC and TPH)
The potatoes were quickly washed and dried, then cut into small cubes, and about 3 g was weighed and extracted with 27 mL of methanol:water (9:1, by volume) by vortexing followed by sonication of the sample in an ice-water bath (0°C, 15 min). The mixture was centrifuged in a Thermo IEC Multi/RF with an 8850 rotor (Thermo Fisher Scientific Inc., Waltham, MA) at 20,000g for 30 min at 4°C, and the aspirated supernatant was stored at À80°C. The supernatants were evaluated by the ABTS (2,2′-azino-bis[3-ethylbenzothiazoline-6-sulfonic acid]) and TPH methods.

Extraction (HPLC)
The potatoes were quickly washed and dried, then cut into small cubes, and about 50 g was weighed, frozen at À20°C, and lyophilized for 3 days. The dry structure was grounded and kept ready for ethanol/water extraction. One gram of each lyophilized sample was extracted with 10 mL of methanol:water (9:1, by volume) by vortexing, followed by sonicating the sample in an ice-water bath (0°C, 15 min). The mixture was centrifuged at 20,000g for 30 min at 4°C, and the aspirated supernatant was stored at À80°C. The supernatants were hydrolyzed and evaluated by HPLC. . Colored potatoes collected in Bolivian Altiplano. Samples 1, 2, and 3 belong to "Ñojchajachaya" cultivar, samples from 4 to 10 belong to "Caty" cultivar, 11 belongs to "Chocapito-morado" cultivar, 12 belongs to "Ajanwiri (clon)" cultivar, and 13 belongs to "Durazno Imilla 30 CN-010" cultivar.

TAC evaluated by the ABTS and TPH methods
Folin-Ciocalteu reagent, gallic acid, sodium carbonate, and acetone were purchased from Merck (Darmstadt, Germany). ABTS, potassium persulfate, Trolox (6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid, 97%), and TPTZ (2,4,6-tripyridyl-s-triazine) were obtained from Sigma-Aldrich (St. Louis, MO), ferric chloride from ICN Biomedicals (Costa Mesa, CA), glacial acetic acid and sodium acetate from BDH Chemicals (Poole, U.K.) and methanol from Laboratory Supplies (Poole, U.K.). All chemicals were of analytical grade. To oxidize the colorless ABTS to the blue-green ABTS + radical cation, 5 mL of ABTS solution (7 mmol/L) was mixed with 88 lL of K 2 S 2 O 8 (140 mmol/L) and stored at room temperature in the dark overnight. On the day of the analysis, the ABTS + radical cation solution was diluted with acetate buffer to reach an absorbance of 0.70 (AE0.02) at 734 nm. A Trolox standard stock solution, 5 mmol/L in ethanol, was diluted with acetate buffer to concentrations of 20-200 lmol/L. The extraction follows Nilsson et al. (2005) using modifications suggested by Peñarrieta et al. (2008). Different standards or samples (100 lL) were added to 1 mL of ABTS + solution, mixed for 30 sec, after which the absorbance reading was started after another 30 sec and maintained for 6 min at 734 nm and 25°C. The concentration was plotted against percent inhibition, which was used for the calculation. The results are expressed as lmol Trolox equivalents per gram of dry matter (lmol TE/g dm).
Total phenolic compounds were measured by the Folin-Ciocalteu reagent which oxidizes the phenolic compounds to phenolates at alkaline pH in a saturated solution of sodium carbonate, resulting in a blue molybdenum-tungsten complex (Singleton and Rossi 1965). However, the method has a limited specificity and a number of substances (particularly ascorbic acid and fructose) (Prior et al. 2005) can be assumed to contribute to the apparent result (Lachman et al. 2008a). Lachman et al. (2008) analyzed the interference systematically and provided data of reaction absorbances of potentially interfering substances. Using these data and data on fructose (Mano et al. 2007), the interference can be estimated to be less than 5%.
The Folin-Ciocalteu reagent was diluted with water (1:10 by volume) prior to analysis. A gallic acid stock solution was prepared in 80% aqueous acetone (1:1 by volume), and the gallic acid standard curve was diluted with water to concentrations of 235-1180 lmol/L. From each standard solution and sample, 50 lL was mixed with 2.5 mL of Folin-Ciocalteu reagent and 2.0 mL of sodium carbonate solution. The samples were mixed and incubated at 45°C for 30 min. The absorbance was read at 765 nm after cooling at room temperature. The absorbance of each sample was compared with the values obtained from a calibration curve using gallic acid as standard. The results are expressed as lmol gallic acid equivalents per gram of dry matter (lmol GAE/g dm) (Peñarrieta et al. 2008(Peñarrieta et al. , 2009.
Before analyzing the samples in HPLC, they were hydrolyzed by refluxing 250 lL methanol:water (9:1) extracts with 250 lL 3 N HCl and 250 lL of baicalein (internal standard) for 1 h at 90 C.
Most of the polyphenolic compounds are bound to insoluble polysaccharide components and the hydrolysis is used to release polyphenols from their corresponding glycosides.
The chemical instability during extraction and analyses of anthocyanidins is a particular concern (Reyes et al. 2004). To protect the components the samples were stored frozen, lyophilized, and the HPLC was run directly after the hydrolysis (the acid condition also stabilizes the anthocyanidins) (Fuhrman 2001;Reyes et al. 2004). The HPLC analysis was made in methanol-water extracts from lyophilized samples.
The polyphenolic compounds were separated using an Agilent liquid chromatographic system comprising a vacuum degasser (G1322 A), a solvent delivery system (Quat Pump-G1311A), an auto-injector ALS (ALS-G1329A), a column oven (ColCom-G1316A), and a diode array detector (G1315B) (Agilent, Santa Clara, CA). The column was a reverse-phase Agilent eclipse plus C18 (150 9 4.6 mm) protected by a 10 mm precolumn. The flow rate was 0.8 mL/min and the injection volume was 20 lL. The mobile phase was a binary solvent system consisting of (A) 1% acetic acid/water and (B) methanol and the gradient used was 40% B at 0 min, 65% B after 5 min, 90% B after 10 min, and 40% B after 15 min until 17 min. The UV absorbance of the eluate was recorded using a multiple diode array detector (190-550 nm). Retention times and absorbance spectrum profiles were compared with standards. Pure standards were also added to the samples as control and peak splitting was used to confirm the identification (Carrasco et al. 2011;Peñarrieta et al. 2011). The samples were quantified at 310 and 530 nm using baicalein as internal standard and using pure standards for specific absorbance determination (Peñarrieta et al. 2008).
DNA was isolated from 50 mg of lyophilized sample of purple potatoes. Around 50 g of fresh sample was frozen at À20°C and had been lyophilized by LabconcoFreeZone 4.5 that reach À50°C (Labconco, Kansas, MO) and grounded into powder in mortar. A DNeasy Plant Mini extraction kit (Qiagen GmbH) was then used for DNA isolation (according to the manufacturer's instructions). The DNA quality was verified by electrophoresis on 1% agarose gel and quantified using a spectrophotometer.

Polymerase chain reaction
Polymerase chain reaction (PCR) amplification was made using 40 ng of DNA and Master Mix (Promega), with the following concentrations: 0.2 mmol/L dNTPs, 1.5 mmol/L MgCl 2 , 0.03 l/lL Taq DNA polymerase (Promega), and 1 lmol/L for primers (forward and reverse), respectively. Thermal cycling conditions were found by BIOER thermocycler for 10 primers referred ) those which are on a range of 400-900 bp: bch6, 21ba, ugp-ase, ans, ct-203, chi, and gp-24 and the following conditions: 3 min denaturation at 95°C, 1 min annealing in the range of 48-57°C and 1 min, 30 sec extension at 72°C, and the number of cycles was between 30 and 33. For primers in the range 1000-1600 bp: stan1, chs, and zep, the conditions were 2 min denaturation at 95°C, 1 min annealing in the range 62-65°C and 1 min, 30 sec extension at 72°C, and 35 cycles. The products obtained after PCR optimization were evaluated by horizontal electrophoresis in agarose gel (2%), stained with ethidium bromide (0.1 lL/mL; Promega), and visualized with a ultraviolet light transilluminator with the addition of a molecular weight marker 100 to 3000 bp (100-bp DNA ladder; Solis Biodyne).

Sequence analysis
Phylogenetic trees were made based on ans and stan1 genes. Primers reported by Zhang et al. (2009) were used to amplify partial regions of ans and stan1. Genes and the amplicons were purified using Qiagen DNA Cleanup Systems and sequenced. Raw sequences were imported in Geneious Pro (Ver. 5.4.6, Biomatters Ltd., Auckland, New Zealand) and homology searches were done by the Basic Local Alignment Search Tool (BLAST) to identify protein-coding regions (exons). Exon sequences were imported in MEGA 5 and aligned using multiple sequence alignment -CLUSTALW (Tamura et al. 2011). The phylogenetic trees were built with the maximum composite likelihood model and the neighbor-joining method (Tamura et al. 2011). Bootstrap analysis was conducted with 1000 replicates. The Ans phylogenetic tree was constructed based on the following genes: ans10, ans12, ans7, ans6, ans4, ans5 (GenBank accession number: KC752436), ans9, and ans8. It was divided in three clades called B1, B2, and B3 originating from a common ancestor, the same for stan1 phylogenetic tree, respectively. The stan phylogenetic tree was constructed based on the following genes: stan12, stan9, stan3, stan1, stan2, stan10 (GeneBank accession number: KC752437), stan6, stan8, stan5, and stan11. It was divided in three clades called A1, A2, and A3 originating from a common ancestor.

Statistical analysis
The principal component analysis (PCA) was performed using MATLAB Release 2012a (MathWorks, Natick, MA).

Results and Discussion
Chemical composition of different cultivars TAC and TPH of colored potatoes are shown in Figure 3, ranging from 1.0 to 7.0 lmol Trolox/g dry weight (dw) and 8.0-17.0 lmol GAE/g dw, respectively. The TAC values obtained are similar to those reported in the literature for potato (1 lmol Trolox/g dw) ) as shown in Table 1. The TPH results are similar to those reported previously for potatoes (10 lmol GAE/g dw) ) as well as of purple-fleshed potatoes (19.3 lmol GAE/g dw) (Lachman et al. 2008a,b) in Stachy locality of Czech Republic with environmental conditions similar to that in the Andean region (lower temperature and higher altitude; see Table 1).
The polyphenols were identified and quantified (Table 2) by comparison with standards using HPLC with a UV-Vis diode array detector. Seven individual compounds were identified: cyanidin, pelargonidin, ferulic acid, caffeic acid, chlorogenic acid, gallic acid, and syringaldehyde (Figure 4). Baicalein was used as the internal standard ).
The absorption maxima for cyanidin and pelargonidin were found at 530 nm and for ferulic acid, caffeic acid, chlorogenic acid, gallic acid, and syringaldehyde at 310 nm. The presence of pelargonidin and cyaniding has been reported in Shetland Black cultivar purple-flesh potato by Lachman et al. (2012). Also, the polyphenols identified in this study have been reported before in purple-fleshed potatoes (Friedman 1997;Lewis et al. 1998;Del Mar Verde M endez et al. 2004).
The quantitative data are summarized in Table 2. The amount of individual polyphenolic compound varies between cultivars. Gallic acid is the main polyphenolic compound in all samples followed by chlorogenic acid and caffeic acid, while ferulic acid is present at lower concentrations. The caffeic acid concentration is similar among the samples, while cyanidin and pelargonidin levels are higher in some of the "Ñojchajachaya" samples (Table 2).
Pelargonidin values obtained (0.004-0.07 lmol/g dw) were comparable to that found in the Shetland Black cultivar purple-flesh potato (0.02 lmol/g dw) (Lachman et al. 2012), but the results were lower than those reported in the literature for red potato (2.3-23.3 lmol/g dw) (Lewis et al. 1998). Also, cyanidin values (0.003-0.07 lmol/g dw) were comparable to that found in Shetland Black cultivar (0.01 lmol/g dw), which was the only purple-flesh potato with both anthocyanidins reported (Lachman et al. 2012).
The ferulic acid content ( On the other hand, lower values were obtained for chlorogenic acid content in this study (0.04-0.5 lmol/g dw) compared with literature data (1 lmol/g dw) (Del Mar Verde M endez et al. 2004;Peñarrieta et al. 2011).
The values for gallic acid (1.2-7.6 lmol/g dw) are slightly higher than previously reported data (1.1 lmol/g dw). The values for syringaldehyde (0.01 lmol/g dw) are similar to values reported previously .
PCA was used to reveal potential patterns between the 13 colored potato samples and TAC, TPH, concentrations  (Lachman et al. 2008(Lachman et al. , 2008. 3 TAC evaluated by ABTS method and TPH by Folin-Ciocalteu reagent .   of individual anthocyanidins and polyphenolic compounds are the nine measured parameters. The explained total variance of PC1 is 32% and of PC2 is 32% (Fig. 5).
The "Nojchajachaya" group is scattered along the second PC, with some samples displaying a high concentration of cyanidin, pelargonidin, and syringaldehyde, whereas one sample displays low concentrations. These samples also have similar levels of TAC (Fig. 3). The Caty group is scattered around the center of the coordinates and therefore does not display any pattern. The results show that the botanical classification only describes limited aspects of the variability of this material.

Analysis of genes associated with the biosynthesis of anthocyanidin
In the beginning about 10 genes associated with anthocyanidin were analyzed (Fig. 6). However, only three (chi, ans, and stan1) of the 10 genes are directly associated with the biosynthesis of anthocyanidin in colored potato. The three genes, chi, ans, and stan1, were present in all individual samples as confirmed by gel electrophoresis (Fig. 6). The three genes of each sample were sequenced. The size of the fragments (500 bp) of the chi gene material was too small to allow for a proper phylogenetic analysis. The phylogenetic trees were made for ans (800 bp) and stan1 (1200 bp) sequences. For some samples, individual samples had to be excluded due to poor resolution; for the ans gene, only eight could be used and for the stan1 gene only 10 could be used of the 13 individual samples.
The stan1, from here on only termed stan, phylogenetic tree clearly shows how samples of the stan gene are grouped together, how they originate from the same node, and that they are completely separated from Nicotiana tabacum, thereby confirming the uniformity in the material making contamination unlikely (Fig. 7).
The stan phylogenetic tree is divided into three clades by common nodes. The genes stan12 (i.e., the stan gene from sample 12), stan9, stan3, stan1, stan2, and stan10 are grouped in the same clade called A1 and they have 88% similarity. In clade A2 the genes stan6, stan8, and stan5 are branched together with a common node with a similarity above 98%. Clade A3 (stan11) shows 83% similarity with clades A1 and A2. The ans phylogenetic tree can be divided into three clades (B1, B2, and B3) that are grouped together and are well separated from N. tabacum and Gentiana triflora (Fig. 8). In clade B1 there are genes from thee samples ans10, ans12, and ans7, which belong to a common node with a 62% of similarity. Clade B2 is formed by ans6, ans4, ans5, and ans9, with a common node, and clade B3 is formed by ans8 with 55% similarity to clades B1 and B2.
The explanatory power of the analysis of the ans genes of samples may reflect the larger biological variation in the ans gene compared to the stan gene.
The botanical classification is based on morphological characteristics of the tubers (Table 3), and the stan and ans phylogenetic trees (Figs. 7,8) show no direct similarity as the different cultivars are not grouped in the phylogenetic trees.

Correlation between phylogenetic trees and chemical composition
PCA was used to reveal patterns among clades from stan and ans phylogenetic trees and the chemical composition of individual samples. The PCA of the eight stan samples gave PC1 and PC2 together explaining 64% of the total variance (Fig. 9). The three clades A1, A2, and A3 enclose each other. Thus, this result shows that the variance in the chemical composition is not explained by differences in the stan gene. Figure 10 shows the corresponding PCA for the ans gene for the eight samples where PC1 and PC2 together explain 71% of the variance. Clades B1 and B2 are from two separated areas, indicating that the ans gene has a clear impact on the chemical composition. Clade B1 has a correlation with a somewhat higher level of pelargonidin. B2 is located along the positive PC1 and thereby correlates with high levels of ferulic acid, gallic acid, caffeic acid, chlorogenic acid, and TPH (Fig. 10).
The ans gene is directly involved in anthocyanidin synthesis. The anthocyanidin has a more final position in the biochemical pathways (Singleton and Rossi 1965;Nilsson et al. 2005;Mano et al. 2007;Peñarrieta et al. 2008;Davies et al. 2009).
The stan gene controls dihydroxyflavonol-4-reductase activity (Bai et al. 2011;Lachman et al. 2012) that leads to the formation of leucoanthocyanins that may be converted into anthocyanidins or into cathechins and condensed lignins (Fig. 1). Thus a less clear connection can be assumed. Thus potato color is confirmed as a sign not only of high level of anthocyanidins but also of polyphenols in general (Fuhrman 2001).
Naturally, not only the genetics are expected to determine the chemical composition as other aspectsextrinsic factors: environmental conditions, cooler temperatures (Reyes et al. 2004) growing conditions (Lachman et al. 2008a;Hamouz et al. 2010)and the intrinsic factor: the cultivar (Andre et al. 2007;Hamouz et al. 2010)may play a role. However, the samples in this article were collected at an experimental farm with uniform growing conditions, which should limit the influence of extrinsic factors on the results.