Characterization and Transcriptional Profile of Genes Involved in

Article pubs.acs.org/JAFC

Characterization and Transcriptional Profile of Genes Involved in Glycoalkaloid Biosynthesis in New Varieties of Solanum tuberosum L. Roberta Fogliatto Mariot,† Luisa Abruzzi de Oliveira,‡ Marleen M. Voorhuijzen,§ Martijn Staats,§ Ronald C. B. Hutten,∥ Jeroen P. van Dijk,§ Esther J. Kok,§ and Jeverson Frazzon*,† †

Food Science Institute and ‡Department of Cellular and Molecular Biology, Federal University of Rio Grande do Sul (UFRGS), Porto Alegre 90050-170, Brazil § RIKILT Food Safety Institute, Wageningen University, 6708 PB Wageningen, The Netherlands ∥ Plant Breeding, Wageningen UR, 6708 PB Wageningen, The Netherlands ABSTRACT: Before commercial release, new potato (Solanum tuberosum) varieties must be evaluated for content of toxic compounds such as glycoalkaloids (GAs), which are potent poisons. GA biosynthesis proceeds via the cholesterol pathway to α-chaconine and α-solanine. The goal of this study was to evaluate the relationship between total glycoalkaloid (TGA) content and the expression of GAME, SGT1, and SGT3 genes in potato tubers. TGA content was measured by HPLC−MS, and reverse transcription quantitative polymerase chain reactions were performed to determine the relative expression of GAME, SGT1, and SGT3 genes. We searched for cis-elements of the transcription start site using the PlantPAN database. There was a relationship between TGA content and the relative expression of GAME, SGT1, and SGT3 genes in potato tubers. Putative promoter regions showed the presence of several cis-elements related to biotic and abiotic stresses and light. These findings provide an important step toward understanding TGA regulation and variation in potato tubers. KEYWORDS: Solanum tuberosum, glycoalkaloids, GAMEs, SGT1 and SGT3 genes, gene expression

■

INTRODUCTION

Despite the significance of GAs, studies on their biosynthetic pathway and the factors that regulate GA levels were scarce until recent years. It is known that GAs are produced in all parts of the potato plant, including the leaves, roots, tubers, and sprouts, and that biosynthesis proceeds via the cholesterol pathway in the following steps: acetate (C2) → mevalonate (C6) → isopentenyl pyrophosphate (C5) → squalene (C30) → cholesterol (C27).1,12 However, many steps between cholesterol and the formation of α-chaconine and α-solanine are unknown. Up to 2013, only enzymes in the final steps of the pathway from cholesterol to GA, such as solanidine galactosyltransferase (SGT1), solanidine glucosyltransferase (SGT2),13−15 and βsolanine/β-chaconine rhamnosyltransferase (SGT3)16 had been described. Itkin and collaborators described the glycoalkaloid metabolism (GAME) genes and proposed a GA biosynthesis pathway.17 Research has been more focused on tomato (Solanum lycopersicum) rather than potato, and just a few aspects are still not elucidated. Since the nature and concentration of glycoalkaloids are genetically determined, there is a strong influence of biotic and abiotic factors on the amount produced by the plant, and therefore, this is particularly noticeable by the difference in gene expression. The aim of the study was to evaluate a possible relationship between TGA content and the expression of GAME, SGT1, and SGT3 genes in edible tubers of potato with different TGA contents. In addition, the putative promoter regions of

Potato (Solanum tuberosum L.) is the fifth most important human food crop in the world after rice, wheat, maize, and sugar cane. The introduction of new varieties of potatoes for consumption or food processing requires analysis not only for pronutritional substances, but also for toxic compounds such as glycoalkaloids (GAs). Moreover, in addition to glycoalkaloids, potatoes contain other biologically active compounds (calystegine alkaloids, antioxidative phenolic compounds, chlorophyll, protease inhibitors, lectins, vitamins) as well as processinginduced browning compounds and acrylamide.1 More than 80 different GAs have been identified in various potato species. The two major GAs in cultivated potatoes are α-chaconine (ca. 60%) and α-solanine (ca. 40%),2−4 which usually comprise more than 95% of the total GA content.5 These are steroidal glycosides derived from aglycon solanidine that vary in their trisaccharide sugar content and are important natural toxic components of potato tubers.6 GAs are potent poisons with a lethal dose of 3−5 mg·kg−1 of body weight, similar to that of strychnine and arsenic.7 The symptoms of GA poisoning include gastrointestinal disorders, confusion, hallucinations, partial paralysis, convulsions, coma, and even death.8 Because GAs are not destroyed during cooking or frying,9 an upper limit of 20 mg/100 g of fresh weight of potato tubers has been established for safe release of new commercial varieties.10 The highest concentration of total glycoalkaloids (TGAs) within a tuber is found in the skin, just beneath the skin surface (up to 1.5 mm deep), and within the eyes and damaged areas.6,10 TGA content varies greatly among individual tubers of the same potato genotype.11 © XXXX American Chemical Society

Received: November 20, 2015 Revised: January 13, 2016 Accepted: January 14, 2016

A

DOI: 10.1021/acs.jafc.5b05519 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry Table 1. Field Information and Total Glycoalkaloid Content of the Eight Potato Samples Used in This Study sample ID

a

varieties

RH00-2

RH00-386-2

RH386-1 RH-029-2

RH00-386-2 RH4X-029-2

RH29-2 HZ-2

RH4X-029-2 HZ94DTA11

HZ94-2 RH036-1

HZ94DTA11 RH4X-036-11

RH36-1

RH4X-036-11

grandparents

year of harvest

time postharvest (days)

TGA contenta (mg·kg−1)

RH97-649-11 × 96-2039-10

IVP92-057-17 × SPG 15458-B18 (Solanum spegazzinii), RH89-050-25 × RH89-035-38

2011

13

396.94 ± 54.12

M 94-110-2 × FRIESLANDER

93-71-3 (Solanum hougasii) × W 72-38-720, GLORIA × 74 A 3

2012 2011

28 13

226.73 ± 30.91 386.93 ± 52.76

RH90-012-2 × RH89-039-16

RH87-217-34 × TAR 24717-4 (Solanum tarijense), BC 1034 × SUH 2293

2012 2011

28 13

296.23 ± 40.39 79.48 ± 10.84

M 94-125-1 × FRESCO

BILDTSTAR × 93-114-5 (Solanum fendleri), CEB 60-15-28 × PROVITA

2012 2011

28 13

22.11 ± 3.01 58.87 ± 8.03

2012

28

63.03 ± 8.59

parents

Sum of α-chaconine and α-solanine ± standard deviation. in duplicate, followed by addition of an extraction solution (acetonitrile/water/formic acid, 50:50/0.2), homogenized with a rotatory tumbler for 60 min, and centrifuged at 4000g for 15 min. After separation by centrifugation, 1 mL of the supernatant was transferred to two different tubes and kept at −20 °C until analysis. On the day of analysis, 50 μL of the supernatant was transferred to four new tubes already filled with an acetonitrile and water solution (50:50), called the “sample solution”, in the following quantities: tube 1, 950 μL; tube 2, 925 μL; tube 3, 900 μL; tube 4, 850 μL. Each tube was analyzed by liquid chromatography−mass spectrometry (LC-MS), using spiking solutions to compare the results. RNA Isolation and Quality Assessment. RNA was isolated from 0.5 g of each freeze-dried sample, according to the hexadecyltrimethylammonium bromide (CTAB) buffer lysis method, followed by chloroform/isoamyl alcohol extraction and overnight precipitation with lithium chloride (LiCl) with some modifications,20 as follows: Lysis was performed with the extraction buffer prewarmed to 60 °C before use, the chloroform/isoamyl alcohol extraction was repeated three times before the LiCl precipitation, and the final precipitation with 96% ethanol was performed with the tubes kept on ice and then centrifuged at 4 °C for 15 min at 14000g. Total RNA isolated was dissolved in 100 μL of 10 mM Tris (pH 7.0) and warmed to 65 °C for 10 min. Total RNA was stored at −80 °C until use. The RNA purity and concentration were assessed by absorbance measurements using a NanoDrop Lite spectrophotometer. For integrity evaluation, 1 μg of RNA was migrated by electrophoresis (50 min at 90 V) in denaturing agarose gel (1.5% agarose, 1× TBE) stained with ethidium bromide. Complementary DNA Synthesis. For each RNA sample, cDNA was prepared in duplicate, as recommended.21 Initial treatment with the enzyme DNase was carried out to eliminate the contamination of genomic DNA molecules. For this, 1 μg of total RNA from each sample was taken in a total of 10 μL of a solution containing RQ1 RNase-free DNase (Promega), 10× reaction buffer [400 mM Tris−HCl (pH 8), 100 mM MgSO4, and 10 mM CaCl2] (Promega), and diethyl pyrocarbonate (DEPC) (Sigma-Aldrich) treated water. After incubation at 37 °C for 30 min, 1 μL of stop solution [20 mM EGTA (pH 8.0)] (Promega) was also added, with further heating at 65 °C for 15 min. Initiating the synthesis of complementary DNA (cDNA), the second step consisted of adding 1 μL of oligo(dT) 15 primer with a concentration of 0.5 μg/μL that hybridizes to the poly(A) tail of mRNA (Promega), followed by incubation at 70 °C for 10 min, and the third last step was characterized by the addition of a 25 μL reaction mixture with 5 μL of M-MLV reverse transcriptase 5× reaction buffer [50 mM Tris−HCl (pH 8.3 at 25 °C), 75 mM KCl, 3 mM MgCl2, and 10 mM DTT] and 1 μL of 200 U of M-MLV reverse transcriptase (both from Promega), 1.25 μL each of dATP/dCTP/dGTP/dTTP (10 mM) (Invitrogen), 0.65 μL of RNaseOUT recombinant ribonuclease inhibitor (Invitrogen), and DEPC (Sigma-Aldrich) treated

the GAME, SGT1, and SGT3 genes were analyzed to check the presence of cis-elements related to the response of the plant to biotic and abiotic stresses, since unpredictable variations in TGA levels can be triggered by light exposure and stress factors.

■

MATERIALS AND METHODS

Database Search and in Silico Characterization. To identify the glycoalkaloid metabolism (GAME), solanidine galactosyltransferase (SGT1), and β-solanine/β-chaconine rhamnosyltransferase (SGT3) gene locations on the potato genome, gene and cDNA sequences and lengths, exon−intron−exon junctions, transcripts, and all information needed for S. tuberosum were obtained from the Ensembl Plants database (http://plants.ensembl.org/index.html), on the basis of The Potato Genome Sequencing Consortium.18 The putative promoter region from the 2000 base pairs (bp) upstream of the transcription start site of each gene was used to search for putative cis-elements. The analysis was performed using the Plant Promoter Analysis Navigator (PlantPan) (http://plantpan.mbc.nctu. edu.tw/index.php),19 and the cis-elements identified were classified on the basis of their putative biological functions. The structures of potato GAME, SGT1, and SGT3 genes selected for the functional analysis and their alternative transcripts were analyzed. Ethics Statements. The field experiments (normal yield trials) in both years were performed on a trial field in the proximity of Wageningen (GPS coordinates 51.95230, 5.63490) owned by Wageningen UR. No specific permission was required to carry out these potato trials. Field Experimental Design. Eight edible potato tubers in total from four distinct genotypes, breeding clones, obtained in duplicate, one grown in 2011 and the other in 2012, with a postharvest storage time of 13 and 28 days, were cultivated at Plant Breeding Sciences, Wageningen University and Research Center (WUR), Wageningen, The Netherlands. The genotypes HZ94DTA11 and RH00-386-2 are diploid potato breeding clones, and the genotypes RH4X-029-2 and RH4X-036-11 are tetraploid potato breeding clones. Although all four clones have a wild potato species clone as a grandparent, they are all considered and treated as “normal” potatoes (S. tuberosum). All potato samples are listed and detailed in Table 1. Sample Preparation. Four average-sized tubers from different sides of individual tubers were selected to minimize variation effects. The potato tubers were washed in water at room temperature, dried with paper, and chopped using a food processor into 1 cm3 cubes. The potato cubes were immediately frozen in liquid N2 to avoid tuber oxidation, packed in plastic bags, and stored in an ultrafreezer at −80 °C. The samples were sent to ZIRBUS Technology, Tiel, The Netherlands, for lyophilization, milling, and vacuum packaging. Potato powder was stored at room temperature until use. TGA Analysis. The quantification of α-chaconine and α-solanine was performed using 40 ± 0.5 mg of each freeze-dried potato sample B

DOI: 10.1021/acs.jafc.5b05519 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

C

oxidation at C22 and closure of the E-ring result in furostanol-type aglycon

cholesterol is hydroxylated at C22

cholesterol is hydroxylated at C26

cholesterol is hydroxylated at C26 22,26-dihydroxycholesterol is hydroxylated at C16

26-aldehyde substrate for transamination catalysis (nucleophilic attack of the amino nitrogen at C22) galactosyltransferase

GAME6 (glycoalkaloid metabolism 6)a

GAME7 (glycoalkaloid metabolism 7)a

GAME8a (glycoalkaloid metabolism 8a)b

GAME8b (glycoalkaloid metabolism 8b)b GAME11 (glycoalkaloid metabolism 11)a

GAME12 (glycoalkaloid metabolism 12)

rhamnosyltransferase

NA

SGT3 (β-solanine/β-chaconine rhamnosyltransferase)

C2c

SGT1 (solanidine galactosyltransferase)

furostanol-type aglycon oxidized to 26-aldehyde

enzyme function in glycoalkaloid pathway

GAME4 (glycoalkaloid metabolism 4)

gene

PGSC0003DMG400023712

PGSC0003DMG400011740

PGSC0003DMG400011749

PGSC0003DMG400024281

PGSC0003DMG400026586 PGSC0003DMG400011751

PGSC0003DMG400026594

PGSC0003DMG402012386

PGSC0003DMG400011750

PGSC0003DMG400024274

gene code

10: 57539858− 57542161

7: 41719828− 41721552

7: 41953952− 41955645

12: 5776516−5780741

6: 44479607− 44481751 7: 41845573− 41847191

6: 44439443− 44441537

7: 52651099− 52659776

7: 41884815− 41887155

12: 5853429−5858832

location

PGSC0003DMT400060959

PGSC0003DMT400030650

PGSC0003DMT400030670

PGSC0003DMT400030677 PGSC0003DMT400062385

PGSC0003DMT400030676

PGSC0003DMT400068373

PGSC0003DMT400032242 PGSC0003DMT400032232 PGSC0003DMT400032240 PGSC0003DMT400032234 PGSC0003DMT400032241 PGSC0003DMT400068388

PGSC0003DMT400030673 PGSC0003DMT400030672 PGSC0003DMT400030671 PGSC0003DMT400032233

PGSC0003DMT400030674

PGSC0003DMT400062367

transcript code

5′-TGGGCTTCCTTCACTACATC 3′, 5′-CTGGCAAGTGATTGTTCCAATA-3′ 5′-GCTTCACATCCCTCGTATTT-3′, 5′-CTAGATTAGGCTGCTTGTGAG-3′ 5′-GACACTTGGCCTTGCTATT-3′, 5′-TGACGGCCAACTCTATCT-3′ 3′-GGCCACTCAGATTGTCTCTATG-5′,

5′-CCTCCAAGATATAGGGAGGTTATTG-3′, 5′-AAACCTAGCCCTTCAGCTAAC-3′

5′-ATAATGAACATGATCTTCCAAGAGGTG-3′, 5′-GTTCTTTACGGTGCTTCGCATAA-3′

5′-GACACAGGCCAGAGAAGAAG-3′, 5′-GGGATATAGCCGTAATGACTCG-3′

5′-GATCAAATGTTTGTGGAGATTACTGC-3′, 5′-TTCGAGCCTTGAGTCCCTTAT-3′ 5′-GCACGAGGTTCTGAGGTTAT-3′, 5′-CTGTATCGCGATGGATTGTTTG-3′

forward/reverse primer

118

101

101

92

95

75

118

134

100

amplicon length (bp)

88,1

89

87

105.6

98.7

91.0

102.6

99.8

89.0

PCR efficiency (%)

0.998

0.998

0.997

0.996

0.998

0.995

0.996

0.997

0.995

R2

Table 2. Glycoalkaloid Metabolism Genes, Respective Enzyme Function, and Their Reference Genes: Gene Code, Location on the Potato Genome, Transcript (Alternative Splicing) Code, Primer Sequences, Amplicon Length, PCR Efficiency, and Correlation Coefficient (R2)

Journal of Agricultural and Food Chemistry Article

DOI: 10.1021/acs.jafc.5b05519 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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90

0.994

water to the final volume, followed by heating at 40 °C for 60 min. The cDNA was stored at −20 °C until use. Polymerase chain reaction (PCR) end point experiments (MyCycler thermal cycler, Bio-Rad, Hercules, CA) were performed for all samples and for all primer pairs, including samples treated only with the enzyme DNase, to verify whether cDNA synthesis was realized correctly and to confirm the expected amplicon length for each primer pair. Primer Design and Tests. All primers were designed using the Primer Quest tool from IDT DNA (http://www.idtdna.com/ primerquest/Home/Index) with melting temperatures between 58 and 62 °C, GC contents of 45−65%, and amplicon lengths of 75−150 bp. To verify the secondary structures of the primers, Oligo Analyzer software from IDT DNA was used (http://www.idtdna.com/ analyzer/applications/oligoanalyzer/). GAME6, GAME7, and GAME11 genes, and the reference CUL3A gene have alternative splicing; see Table 2. Blasts were carried through to check the common region between different transcripts to design the oligonucleotides. GAME8a and GAME8b genes are copies of each other. They are located on the same chromosome. For this reason, we designed one oligonucleotide for both genes. Consequently, the final relative expression was the sum of the expressions of the genes GAME8a and GAME8b. All primers designed and their respective amplicons for GAME, SGT1, SGT3, and reference genes used in this work for S. tuberosum edible tubers are described in Table 2. To evaluate the PCR efficiency (E), a standard curve was constructed with four points in a 5-fold dilution starting from a 1/5 cDNA concentration (1:5, 1:25, 1:125, and 1:625), in agreement with Perini22 and suggested by Bustin.21 The PCR E and correlation coefficient (R2) were calculated using StepOne software, version 2.3 (Applied Biosystems), on the basis of E = 10−1/slope, slope of the plot Cp (crossing point) versus log input of cDNA. E and R2 for each PCR used in the present work are presented in Table 2. Quantitative PCR (qPCR). All polymerase chain reactions were carried out on a Step One Plus real time PCR system (Applied Biosystems). To monitor the double-stranded DNA synthesis during the qPCR run, SYBR green (BIORAD; 1:10000 dilution) was used. Reactions were performed in 20 μL volumes with 10 μL of diluted cDNA (1:100), a 0.2 μM concentration of the primer pair, 0.1 mM dNTP, 0.25 units of Platinum Taq DNA polymerase (Applied Biosystems), 1× buffer solution (Applied Biosystems), and 1.5 mM MgCl2 (Applied Biosystems). Each cDNA was analyzed in four technical replicates, and negative controls were included for all primers. The following cycles were conducted: 94 °C for 5 min, 40 amplification cycles of 94 °C for 15 s, 60 °C for 10 s, 72 °C for 15 s, and 60 °C for 35 s, and final melting curve between 50 and 99 °C. Δ is 0.3 °C/s. Data Analysis. Data were analyzed by transforming raw Cp, in log 10 (raw Cp) values, into relative quantities using the E−ΔΔCp method.23 Therefore, all data were expressed relative to the expression of the most highly expressed genes identified in a previous study used as reference genes for data normalization.24 The three genes used were C2, exocyst complex component sec3 (SEC3), and CUL3A. E−ΔΔCp (difference between the Ct value of the studied genes and the Ct value of each constitutive gene, generated in total six observations for each sample) values of sample HZ94-2 (lowest TGA content) and seven other different potato samples, for all GAME (GAME4, GAME6, GAME7, GAME8ab, GAME11, and GAME12), SGT1, and SGT3 genes, were determined. For the statistically significant difference (P < 0.05) for sample HZ94-2, compared to the seven other samples, the gene was considered to be more transcribed (E−ΔΔCt > 1) or less transcribed (E−ΔΔCt < 1). Analysis of variance (ANOVA), using SAS software (version 9.3), at a significance level of 5%, was performed to verify whether there was a statistically significant difference between samples with high and low TGA contents. Furthermore, t tests, via SAS software (version 9.3), were performed to verify this set of samples are significantly different from each other.

a

PGSC0003DMT400003338 PGSC0003DMT400003339

PGSC0003DMT400003337 2: 46264503− 46268790 PGSC0003DMG400001321 NA CUL3A (ATCUL3/ATCUL3A/CUL3/CUL3A)a,c

Gene with alternative splicing. bSame sequence and primer. Fifferent code and location on potato genome. cReference gene. NA = not applied.

120

0.995 90.3 75 NA

PGSC0003DMG402015451

12: 56757079− 56759688

PGSC0003DMT400039945

5′-AGCTTTGCTTCTCCTCATACTC-3′ 3′-GGAGCAGTATATCCAAGGACAA-5′, 5′-AGGAACATTGTAGTGACAAACTTAG-3′ 3′-GAGGACCGGTGAAGTGATAAAC-5′, 5′-TCAGCCGAGACATCAAGAAAC-3′ SEC3 (exocyst complex component sec3)c

gene

Table 2. continued

enzyme function in glycoalkaloid pathway

gene code

location

transcript code

forward/reverse primer

amplicon length (bp)

PCR efficiency (%)

R2

Journal of Agricultural and Food Chemistry

D

DOI: 10.1021/acs.jafc.5b05519 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Exon−intron structures of GAME, SGT1, and SGT3 genes generated using software developed by Hu et al.25 The yellow boxes represent the exons, the lines connecting them represent the introns, and blue boxes represent upstream and downstream sequences. Alternative transcripts are also shown.

■

RESULTS AND DISCUSSION Characterization of Exon−Intron Organization and Promoter Regions of GAME Genes. The structures of GAME, SGT1, and SGT3 genes were generated using software developed by Hu et al.25 and are shown in Figure 1. All studied gene codes, isoform/transcript codes, locations on the potato genome, and their functions in the TGA biosynthesis pathway are listed in Table 2. GAME4 is located on chromosome 12 and has one isoform of 1614 bp length, with eight exons, and a length of 7804 bp. In contrast, GAME6 has four isoforms, the first with only one exon, the second and the third with three different exons, and the last with five exons. GAME6 is located on chromosome 7 and has a length of 4741 bp. GAME7 is also located on chromosome 7 and has six isoforms: the first with five exons, the second with three exons, the third with only one exon, the fourth with five exons, the fifth with two exons, and the sixth with four exons. The GAME7 gene is longer than the other GAME genes, with a total length of 11078 bp. The GAME8a and GAME8b genes are copies on the same chromosome; both genes are located on chromosome 6 and have one isoform with five exons. Although copies, the GAME8a and GAME8b genes have differences in the promoter regions and, probably for this reason, a small difference in the gene length, 4495 and 4545 bp, respectively. GAME11, located on chromosome 7, is the shortest gene of the GAME genes, at 4019 bp, with two isoforms both with five exons. GAME12 is located on chromosome 12, has one isoform with 11 exons, and is 6626 bp long.

SGT1 is located on chromosome 7 and has one isoform with one exon and a length of 4095 bp. Finally, SGT3 is also located on chromosome 7 and has one isoform with only one exon and a length of 4125bp. Large and often unpredictable variations in TGA levels can arise from differences in genotype, locality, season, cultivation practice, and stress factors,26 resistance to viral and bacterial diseases,27−29 insect deterrence,30,31 defense response against fungal pathogens,32,33 and harvest and postharvest treatments, such as drought,34 high temperature,35 light exposure of the tubers,33,36 and wounding.37,38 Analysis of the putative promoter regions of the genes involved in GA biosynthesis identified enrichment of putative cis-elements that are associated with the response of the plant to abiotic and biotic stresses.19 Several regulatory cis-elements that are known to be responsive to a variety of stress factors and light were found in GAME, SGT1, and SGT3 gene promoters (Table 3). Three different cis-elements specific to S. tuberosum were also identified. Two light-regulated cis-elements were identified on all analyzed genes: GATABOX contained up to 31 copies on GAME12 in the promoter region, and GT1CONSENSUS contained up to 28 copies on GAME6 in the promoter region. Some cis-elements related to stress were also found on all studied genes. Three were responses to dehydration and water stress: MYB2CONSENSUSAT contained up to 5 copies on GAME12 in the promoter region, MYBCORE presented 5 copies on SGT1, and MYCCONSENSUSAT contained up to 26 copies on GAME8a (Table 3). A further cis-element common to all GAME and SGT3 genes was WBOXATNPR1 for disease response with up E

DOI: 10.1021/acs.jafc.5b05519 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Table 3. Number of Copies and Biological Function of the Putative cis-Elements Related to Stress and Light-Regulated That Were Identified in the GAME, SGT1, and SGT3 Gene Promotersa cis-element ABRE-like ABREATCONSENSUS ABRELATERD1 ABREZMRAB28 ACGTATERD1 ASF1MOTIFCAMV CACGTGMOTIF CBF2 EMBP1TAEM GATABOX GBF1/2/3 GT1CONSENSUS HDZIP2ATATHB2 HSEs IBOX LTRECOREATCOR15 MYB1AT MYB1LEPR MYB2AT MYB2CONSENSUSAT MYBCORE MYBST1 MYCATERD1 MYCATRD22 MYCCONSENSUSAT P1BS SORLIP1AT SORLIP2AT SREATMSD SURE2STPAT21 TAAAGSTKST1 TBOXATGAPB UP1ATMSD WBOXATNPR1 a

function dehydration response and lowtemperature response, water and thermal stress stress-regulated response dehydration response water stress response and cold tolerance response to dehydration abiotic and biotic stress defense-related water stress response various stress treatments light-regulated hypoxia light-regulated light-regulated heat stress light-regulated cold response, thermal stress dehydration response, water stress defense-related water stress dehydration response, water stress water stress unknown function, specific for S. tuberosum dehydration, water stress water stress, dehydration dehydration response, water stress phosphate starvation light-regulated light-regulated injury unknown function, specific for S. tuberosum unknown function, specific for S. tuberosum light-regulated injury response disease response

GAME4

GAME6

GAME7

GAME8a

GAME8b

GAME11

GAME12

1

1

1

1

1 2 2

1 1 4

2

12 1 8

6

6 3

4 2

4

1

1 15

24

19

25

24

22

26

28

22

23 1

26 1 2

19

1 3

1 2

2 2

2 1 4

2 3 1

2 4 2 4

14

12

1 1 2 4 1 1 1 4

5 1

2 2

2 3 2

1 2

2 2 26 4 2 1 1

2 2 14 4

1

15 1 3 3 4 1

12

4 1 4 2 1 31 2 18 1 2 1 3 2 2 5 1 4

SGT1

SGT3

1

1

4 1

4

12

16

22

20

2

1

2 5 2

1 1 1 1 1 1 20

4 4

1

2 4 22

7

6

5

10

11

1

1

3

2

2

7

5

5

2 1

1

8

8

6

2 1 1

2

1

3

4

7

1 1

6

Analysis of a 2000 bp region upstream of the transcription start site of each gene was performed using the PlantPan database.19

Expression Profile of Genes Related to GA Biosynthesis. The efficiencies (E) of primer pairs designed for all genes were evaluated using a standard curve with serial dilutions of S. tuberosum edible tuber cDNA. The correlation coefficient (R2) for all resulting amplification curves was greater than 0.99, and all eight primer pairs allowed amplification efficiencies between 87% and 105.6% (Table 2). Considering that the optimal PCR efficiency is 100%, when the whole target cDNA would be duplicated at every PCR cycle during the exponential phase, the efficiency values obtained were therefore considered acceptable. Hence, the amplification products of each reaction were comparable. The expression of GAME4, GAME6, GAME7, GAME8a, GAME8b, GAME11, GAME12, SGT1, and SGT3 was analyzed by reverse transcription qPCR (RT-qPCR) to verify differences in the expression pattern in edible potato tuber samples with different TGA contents (Figure 2B,C). To normalize the expression profile of those genes in different potato samples, the lowest TGA content sample was used to normalize the

to seven copies in the GAME8b promoter region (Table 3). These results may indicate that those genes respond to these stress conditions. TGA Content. The eight potato samples from four different genotypes were divided into two groups according to their glycoalkaloid content. The first group was composed of genotypes RH00-386-2 and RH4X-029-2, which presented a high TGA content in the range from 226.73 to 396.94 mg·kg−1. The second group comprised samples with a low TGA content (HZ94DTA11 and RH4X-036-11 genotypes) in the range from 22.11 to 79.48 mg·kg−1 (Table 1). The TGA content in tubers of table potatoes generally ranges from 20 to 100 mg·kg−1, resulting in only a small safety margin for consumers at normal levels of potato consumption. Unfortunately, it is not rare that the upper safe level of TGA in potato tubers is surpassed. Some cultivars such as Lenape, Magnum Bonum, and Ulster Chieftain have been withdrawn from some countries’ markets due to frequently exceeding the upper safe limit of 200 mg·kg−1 for total glycoalkaloid content.39 F

DOI: 10.1021/acs.jafc.5b05519 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Desirée via the independent downregulation of SGT genes with promising results. However, transgenic plants still revealed other significant (unintended) differences between the SGT line and the wild type, and also between the wild type and SGT lines used. Glycoalkaloids are cited as toxic in the Consensus Document on Compositional Considerations for New Varieties of Potatoes, and information on GA content is needed for use during the regulatory assessment of new potato varieties.26 Due to the significance of GAs for food safety, knowledge of the expression and regulation of genes that participate in the GA biosynthetic pathway in potato tubers may provide an important insight into GA prediction and control.42 In conclusion, an analysis of the putative promoter regions of GAME, SGT1, and SGT3 genes found the presence of several cis-elements related to the response of potato plants to biotic and abiotic stresses and light, as well many copies of ciselements on their promoter regions that confirmed that unpredictable variations in TGA levels could be related to these stressors. There was a relationship between TGA content and the expression of genes involved in GA biosynthesis in potato tubers. These findings provide an important step toward understanding TGA regulation and variation in potato tubers.

Figure 2. Overview of the GA biosynthesis route and a heat map showing the difference in gene expression among two groupshigh and low TGA contents: (A) TGA biosynthesis pathway, (B) heat map showing the expression of GAME, SGT1, and SGT3 genes along TGA pathway in potato tubers with high and low contents of TGA, (C) relative expression of GAME, SGT1, and SGT3 genes in potato tubers with high and low contents of TGA.

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transcript levels in other samples for each gene. As listed in Table 4, all analyzed genes had higher relative expression levels

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +55 51 3308 7115. Fax: +55 51 3308 7048.

Table 4. Relative Expression of GAME, SGT1, and SGT3 Genes in Potato Tubers with High and Low Contents of TGA relative expression gene GAME4 GAME6 GAME7 GAME8a and GAME8b GAME11 GAME12 SGT1 SGT3 a b

high-TGA samples

Funding

We gratefully acknowledge the National Council of Research of Brazil (CNPq No. 300912/2012-9) and Coordenaçaõ de ́ Superior (CAPES) of Aperfeiçoamento de Pessoal de Nivel Brazil for funding this research.

a

low-TGA samples

p

1.027 1.027 0.987 1.039

± ± ± ±

0.017 0.023 0.026 0.020

1.001 0.988 0.976 0.989

± ± ± ±

0.005 0.019 0.041 0.021