ARTICLE pubs.acs.org/jpr
Analysis of Natural Variation of the Potato Tuber Proteome Reveals Novel Candidate Genes for Tuber Bruising Claude Urbany,* Thomas Colby, Benjamin Stich, Lysann Schmidt,† J€urgen Schmidt, and Christiane Gebhardt Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany
bS Supporting Information ABSTRACT: Potato (Solanum tuberosum) presents a challenging organism for the genetic and molecular dissection of complex traits due to its tetraploidy and high heterozygosity. One such complex trait of high agronomic interest is the tuber susceptibility to bruising upon mechanical impact, which involves an enzymatic browning reaction. We have compared the tuber proteome of two groups of 10 potato cultivars differing in bruising susceptibility to (i) identify de novo proteins that contribute to bruising, based on differential protein expression, and (ii) validate these proteins by combining proteomics with association genetics. The comparison of 20 potato varieties yields insight into the high natural variation of tuber protein patterns due to genetic background. Seven genes or gene families were found that were both differentially expressed on the protein level between groups and for which DNA polymorphisms were associated with the investigated traits. A putative class III lipase was identified as a novel factor contributing to the natural variation of bruising. Additionally, tuber proteome changes triggered by mechanical impact, within and between groups, were monitored over time. Differentially expressed proteins were found, notably lipases, patatins, and annexins, showing remarkable time-dependent protein variation. KEYWORDS: potato, bruising, enzymatic discoloration, candidate genes, class III lipase, natural variation, proteomics, association genetics
’ INTRODUCTION Potato (Solanum tuberosum) is one of the most important food crops consumed worldwide and an excellent example of a vegetative propagated, polyploid plant species with complex genetics. The tubers develop on stolons, underground side shoots that enlarge at the tip by cell division and expansion to form a heterotrophic storage organ. Active tuber growth is accompanied by major physiological and metabolic changes that lead to the deposition of large amounts of starch and storage proteins.1 Mechanical impact during harvest, transport, and storage of potato tubers initiates the development of an internal tissue discoloration, commonly referred to as “blackspot bruising”. Part of the latter is an enzymatic browning reaction, which is frequently observed not only in potato tubers but also in fruits and vegetables upon physical damage.26 Another problematic consequence of enzymatic browning is after-cooking darkening (ACD).5,7 Enzymatic browning results from the oxidation of endogenous phenolic compounds and is catalyzed by oxidoreductases, notably polyphenol oxidases (PPOs) or tyrosinases in the presence of oxygen.812 Within damaged cells, oxidized phenolic compounds, particularly quinones, react further in a nonenzymatic fashion and form dark pigments (melanins) attributing a dark gray to black appearance to bruised tissue.8,13 The tissue damage only becomes apparent after removal of the tuber skin. Bruising symptoms vary largely between different r 2011 American Chemical Society
potato genotypes.12 Blackspot bruising is detrimental to food quality and leads to the rejection of the crop by the consumers, retailers and the processing industry resulting in considerable economic losses. Susceptibility to tuber bruising is therefore of high agronomic interest. The enzymatic and structural components affecting tuber bruising (e.g., cell shape, cell number, starch content, membrane stability, and metabolite levels)12,13 depend on multiple genetic factors. In addition to the genetic variation, developmental stage and environmental factors contribute to the phenotypic output of tuber bruising susceptibility. Knowledge of the genes and their natural allelic variants underlying susceptibility to bruising could provide insights into improving the genetic resistance to bruising, either by marker-assisted selection or transgenic approaches. Although the identification of quantitative trait loci (QTL) underlying complex phenotypic traits is feasible, the positional cloning of the causal allelic gene variants remains severely hampered due to the polyploid, noninbred nature of potato.1416 Moreover, the bruising phenotype itself is rather prohibitive for positional cloning due to the cumbersome phenotypic evaluation, requiring large amounts of tubers, which become available only after several years of vegetative multiplication. An alternative to positional QTL cloning is the candidate gene approach, Received: June 30, 2011 Published: November 02, 2011 703
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Table 1. Varieties Selected for Comparative Tuber Proteome Analysis and Their Phenotypic Trait Values (Adjusted Entry Means40) for Bruising Index (BI), Tuber Starch Content (TSC) and SCB (Starch Corrected Bruising) code # Low bruising
High bruising
a
variety
BI
TSC
SCB
3
a
Agila
11.1 ( 5.4
12.4 ( 0.5
14.6 ( 4.2
21 27
Carmonaa Elfea
14.8 ( 5.4 6.9 ( 5.4
15.0 ( 0.5 12.7 ( 0.5
13.0 ( 5.6 13.8 ( 4.7
33
Gala
9.9 ( 5.4
12.5 ( 0.5
24.8 ( 9.8
40
Krone
9.4 ( 5.4
12.8 ( 0.5
0.8 ( 4.4
46
Lolitaa
8.1 ( 5.4
13.8 ( 0.5
7.0 ( 4.4
47
Marabela
7.7 ( 5.4
12.6 ( 0.5
15.6 ( 5.6
66
Quarta
12.5 ( 5.4
14.4 ( 0.5
13.9 ( 13.8
67
Rafaela
12.4 ( 5.4
11.8 ( 0.5
2.1 ( 19.5
69 16
Remarka Aspiranta
12.6 ( 5.4 73.5 ( 5.4
15.1 ( 0.5 19.2 ( 0.5
31.0 ( 13.8 20.1 ( 9.8
19
Calla
56.5 ( 5.4
18.9 ( 0.5
6.2 ( 9.8
32
Fitis
55.4 ( 5.4
17.0 ( 0.5
1.4 ( 9.8
38
Kolibria
72.1 ( 5.4
18.6 ( 0.5
11.0 ( 9.8
41
Kuba
70.6 ( 5.4
19.7 ( 0.5
14.6 ( 4.2
42
Lady Rosetta
65.3 ( 5.4
17.9 ( 0.5
4.6 ( 4.2
45
Logoa
72.7 ( 5.4
21.6 ( 0.5
6.9 ( 4.3
55 60
Maxillaa Olgaa
73.1 ( 5.4 65.7 ( 5.4
20.2 ( 0.5 19.4 ( 0.5
2.0 ( 9.8 11.8 ( 4.3
63
Panda
60.0 ( 5.4
18.6 ( 0.5
49.8 ( 13.8
Varieties used for time course of site-specific tissue damage.
which is based on the knowledge of a gene’s function in controlling a trait of interest and the genetic colocalization of a functional candidate gene with QTL for that trait.17 The candidate gene approach is, however, biased toward genes known to play a role in the trait of interest. Comparative proteome analysis of individuals with contrasting phenotypes provides a means to discover candidate genes de novo, based on differential protein expression. Comparing groups of individuals with contrasting phenotypes (case-control study) reduces the probability of detecting differential proteins unrelated to the phenotypic differences of interest, which result merely from individual genetic background effects. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is a widely used method for separating complex protein mixtures. Using 2D-PAGE and subsequent mass spectrometry (MS) of peptides deriving from tryptic digests of excised protein spots, it is often possible to visualize, quantify, and identify hundreds or even thousands of proteins in a given tissue or cell sample.18 Differentially expressed proteins can not only be the consequence of altered transcription and translation of the coding gene in a specific genetic background but also due to the expression of allelic variants encoding different protein isoforms.19 Even though the number of studies and reports on total potato tuber protein extracts resolved by 2DPAGE is increasing,7,12,18,2028 very little information is available on the natural variation in the tuber proteome.29 Although the potato gene pool used by European breeders has been limited for historical reasons,30,31 cultivars with a large variation of bruising susceptibility and other complex traits have been developed. The introgression of wild Solanum species into the cultivated potato provided a rich source of genetic diversity for breeding purposes and association studies.3239 Association genetics is based on the phenotypic and genotypic analysis of populations of individuals related by descent rather than progeny of a single cross. Recently, we made use of the natural diversity for
bruising susceptibility and other quality traits existing in elite potato varieties and breeding clones. In an association mapping experiment, 205 tetraploid genotypes resulting from breeding programs were evaluated in multiyear and -location trials for bruising susceptibility, tuber starch content, yield, tuber number and shape. The population was genotyped for DNA variation in known as well as novel candidate genes, originating from comparative proteomics. We detected highly significant markertrait associations for bruising susceptibility and other traits.40 In the present paper, we describe the comparative proteome approach which led to the discovery of novel candidate genes that were associated with bruising susceptibility. Two groups of cultivars were selected based on high and low susceptibility to bruising and subjected to proteome analysis based on 2D-PAGE and protein identification by mass spectroscopy. In addition, tuber proteome changes triggered by mechanical impact were monitored in highly bruising resistant and susceptible cultivars. The results are discussed in view of the physiological context of tuber bruising and the present molecular knowledge of the underlying metabolic pathways.
’ EXPERIMENTAL PROCEDURES Plant Material
Based on the phenotypic evaluation of 85 tetraploid varieties for bruising susceptibility,40 ten genotypes each with the highest and lowest bruising susceptibility were selected (Table 1). Field grown tubers of the 20 varieties were provided by EUROPLANT Pflanzenzucht GmbH (EU) and stored for 2 months at 4 °C in the dark. Afterward, tuber tissue without peel was portioned into 50 mL falcon tubes, snap-frozen in liquid nitrogen, freeze-dried and grounded to powder. Freeze-dried samples were stored at 20 °C and used for protein extraction. 704
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were removed by repeating the procedure twice with 1 volume of extraction buffer. Proteins were precipitated by adding 4 volumes of 0.1 M ammonium acetate in 100% methanol to the final phenol phase. After incubation for 1 h at 20 °C, samples were centrifuged (20000 g, 10 min, 4 °C), and the pellets were washed first with 100% acetone and subsequently with an 80% acetone solution. Protein pellets were air-dried and solubilized in 2 M thiourea/7 M urea. Protein quantification was carried out by fluorimetric measurement, using the Qubit system (Invitrogen; Karlsruhe, Germany) according to the instruction of the manufacturer.
Phenotypic Data
Susceptibility to tuber bruising of the 85 varieties has been evaluated according to the guideline from the German Federal Plant Variety Office (Bundessortenamt 30.03.2006) over two years at six locations as described previously.40 The bruising index (BI) was determined, which ranges from 0 (resistant to bruising) to 100 (highly susceptible to bruising). Tuber starch content (TSC) (percent fresh weight) was quantified by measuring specific gravity.40,41 The trait starch corrected bruising (SCB) was derived from the residuals of the regression of BI on TSC.40 Genotyping and Association Analysis
Association analysis was carried out as previously described.40 In addition to the candidate genes and loci presented in Urbany et al.,40 the genes ci21A (U76611) and Annexin p34 (CAB92956) were tested for association with the traits BI, SCB and TSC. Primers for ci21A (foward 50 -GGG CAG GTG GAT TTG CAT TA-30 ; reverse 50 -AAA AAC ACG ACA ACG CTC AG- 30 ) and Annexin p34 (foward 50 - TAT GGA CAC TGG ATC CGT CA30 ; reverse 50 - CGG TAG GAG CTC ACA AGA GG- 30 ) were derived from the accession assigned by mass spectrometry to corresponding protein spots. Amplicons were generated from genomic DNA of the individuals of the association mapping population and subjected to single strand conformation polymorphism (SSCP) analysis.40 Polymorphic SSCP marker fragments were tested for association with the traits BI, SCB and TSC.
Two-dimensional Polyacrylamide Gel Electrophoresis, Image Acquisition and Data Analysis
For analytical 2D-PAGE of the tuber tissue, 150 μg of total tuber protein extract were subjected to isoelectric focusing in the first dimension using the ZOOM IPGRunner System (Invitrogen) according to the instructions of the manufacturer. The rehydration buffer contained 2 M thiourea and 7 M urea instead of 8 M urea. Isoelectric focusing was performed with 7 cm nonlinear IPG strips, pH 310 (Invitrogen) with the recommended Voltage Ramp protocol. The second dimension was run on the XCell SureLock Mini-Cell system (Invitrogen). NuPAGE 412% Bis-Tris ZOOM gels (Invitrogen; 8 8 cm and 1 mm gel thickness) were run in the MES-SDS buffer system at 200 V for 50 min. After the run, gels were stained with Coomassie PAGEBlue (Fermentas, St. Leon-Rot, Germany) according to the manufacturer’s protocols. For proteomic analysis of 350 μg total tuber protein extract by large gels (24 20 cm), isoelectric focusing was carried out using the PROTEAN IEF Cell (BioRad, Hercules, USA) and 24 cm nonlinear Bio-Rad ReadyStrip IPG Strips, pH 310. The four step voltage ramp protocol consisted of an initial step at 250 V for 30 min followed by 2000 V for two hours, 10000 V for three hours and finally 10000 V until reaching 60000 Vhours in total. The second dimension was run on the PROTEAN Plus Dodeca Cell (Bio-Rad). Hand-cast 12% Tris-Glycine gels (24 20 cm; 1 mm thickness) were run in 23 L Tris-Glycine running buffer at 50 V for 30 min, then at a constant voltage of 200 V with no current limit for about six hours until the bromphenol-blue line reached the end of the gels. After the run, gels were fixed in 7% acetic acid/20% isopropanol and stained overnight with Coomassie PAGEBlue (Fermentas, St. Leon-Rot, Germany). Destaining was done by incubating gels in water. Gel images were produced by using a daylight scanner integrated within the Proteineer spII spotting system. Spots were detected by using the default spot detection settings in the Proteomweaver 2-DE analysis software package (Bio-Rad, Hercules, USA). Protein extracts from tubers of each genotype, originating from two consecutive years of field trial constituted two biological replicates. Two technical replicate gels per sample were integrated in the analysis. Gels of genotypes, including biological and technical replicates, with the same bruising phenotype defined a group. Only spots detected in 60% of all replicates within a group were further analyzed and used to create group average gels. Quantification and statistical analysis by MannWhitney U test of the detected spots was also performed with ProteomWeaver.
Targeted Site-specific Damaging
For targeted site-specific damaging, five varieties each with high and low bruising susceptibility, which had shown the most consistent bruising evaluation scores across all environments, were selected (Table 1). Five field-grown tubers per variety were stored for 2 months at 4 °C in the dark and subsequently mechanically damaged by means of an impact pendulum.42 Tubers were damaged around their whole perimeter every two centimeters and orthogonal to the central axis. The impact pendulum had a fixed releasing angle of 50°, a pendulum arm length of 30 cm with a weight of 200 g, and hit the tubers at their perimeter with a flat metal tip with a diameter of 7 mm. After the impact, tubers were immediately cut in half just above the impact zone of the pendulum or after incubation at 4 °C for 30 min, 1 h, 3 and 24 h, and bruising symptoms were documented by photography. Slices of 5 mm width were prepared including the corresponding damaged tissue zone. Then the central tuber flesh was punched out and only the outer rim of the tuber with the skin was frozen in liquid nitrogen. The tissues sampled from five tubers per genotype were pooled, freeze-dried, grounded and subsequently subjected to protein extraction. Protein Extraction
Freeze-dried potato tuber tissue from several tubers per genotype was pooled and ground, using 4 to 5 ceramic beads (5 mm diameter, VWR International GmbH, Germany) per 50 mL Falcon tube and a vortex (Heidolph REAX 2000, Kelheim; Germany). Hereafter 250 mg of ground tissue in a 2 mL reaction tube were vigorously mixed with 750 μL extraction buffer (2% SDS; 0.1% Triton X-100; 10 mM EDTA; 25 mM DTT; 30% Sucrose; 0.05 M Tris/HCl pH 8). After incubating the sample for 30 min on ice, phenol extraction was carried out by adding 1 volume of Tris-buffered phenol (Biomol GmbH, Hamburg; Germany). After phase separation, the upper phenol phase was transferred into another 2 mL reaction tube. Impurities
Sample Preparation for MS Analysis, MALDI Data Acquisition and Database Searching
Protein spots were visually selected and robotically picked from Coomassie stained gels using the Proteineer spII system (Bruker Daltonics, Bremen, Germany). Excised spots were tryptically digested and the resulting peptide mixtures were 705
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Figure 1. Differences of bruising index (BI), tuber starch content (TSC) and starch corrected bruising (SCB) between cases (low bruising genotypes) and controls (high bruising genotypes). Shown are boxplots of the adjusted entry means40 for (A) BI, (B) TSC and (C) SCB between low (Low BI) and high bruising susceptible (High BI) genotypes. Outliers are indicated by open circles. * = groups are significantly different at p < 0.001 as determined by t test, n.s. = not significant.
spotted as an HCCA suspension on AnchorChip TM targets43 using a Proteineer dp robot (Bruker Daltonics). PMF (Peptide Mass Fingerprint) data were collected on an UltraflexIII MALDI ToF/ToF mass spectrometer (Bruker Daltonics) (Supporting Information 1). The spectrometer was calibrated every four samples using a peptide calibrant mix (Bruker catalog number 206195). In addition, tryptic self-digestion peptide masses detected in the spectra were used as an internal standard for postacquisition calibration. PMF spectra resulting from 1200 shots were summed and processed with FlexAnalysis software to produce peak files for searching (see Supporting Information 2). The resulting PMFs were submitted to database searches as described below. Following a first round of database searching and on-target recrystallization of the sample spots, MS/MS spectra (800 shots) were collected on selected precursors as described elsewhere44 in order to confirm PMF-based identifications and elucidate further any unexplained peaks. Both MS and MS/MS data were used to search against the potato unigene builds available at the solgenomics database (http:// solgenomics.net/, SGN-U identifier), the May 2007 release of the NCBI nonredundant database (http://www.ncbi.nlm.nih.gov, genebank entries), and the predicted potato protein data set,45
available at potato genome sequencing consortium web resource (http://www.potatogenome.net, PGSC scaffolds and PGSC protein sequences), using MASCOT (http://www.matrixscience.com) (see Supporting Information 1 and 2). PMF data were searched using a peptide mass tolerance of 50 ppm for postacquisition calibrated spectra and 150 ppm when postcalibration failed. MS/ MS data precursor and fragment ion tolerances were both set to 0.4 Da. Both searches were conducted applying the modifications CAM-cysteine (fixed) and methionine oxidation (variable) and allowing 1 tryptic miscleavage.
’ RESULTS Selection of Cases and Controls for Bruising Susceptibility
European elite germplasm evaluated for BI, TSC and SCB40 was exploited to select two groups of ten tetraploid potato cultivars with contrasting phenotypes, serving as cases (low BI) and controls (high BI). For the cases, BI values ranged from 6.9 ( 5.4 to 14.8 ( 5.4, while the controls showed scores from 55.4 ( 5.4 to 73.5 ( 5.4 (Table 1). The difference between the BI means of the two groups was significant (Figure 1A) (t test, p < 0.001). Tuber starch content was also significantly different between the two contrasting groups (Figure 1B) (t test, p < 0.001). 706
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Figure 2. Exemplary 2D gel of total potato tuber protein extracts. Three-hundred fifty micrograms of protein extracts were separated on a 24 cm IEF strip with a nonlinear pH gradient ranging from 3 to 10 and a 12% Tris-Glycine SDS-PAGE (24 20 cm). Staining and image acquisition was carried out as outlined in experimental procedures. A molecular weight marker is shown on the left of the gel. The migration pattern of the Bio-Rad Precision Plus Protein dual color standard (BioRad, Hercules, CA) is indicated with corresponding molecular weights in kDa.
This is in agreement with previous studies, where a strong positive correlation between tuber starch content and bruising susceptibility has been observed.40,46 Low BI genotypes had a tuber starch content of not more than 15.0 ( 0.5% whereas high BI genotypes were characterized by values ranging between 17.0 ( 0.5 and slightly above 20% (Figure 1B, Table 1). The trait SCB has been calculated from BI and TSC40 to correct for the strong influence of tuber starch content on BI. Interestingly, the two for BI and TSC contrasting groups showed no significant differences for SCB and displayed large withingroup variability (Figure 1C, Table 1). Comparison between the Tuber Proteomes of Cases and Controls
To detect differential tuber protein composition related to bruising susceptibility, total protein was extracted from fieldgrown and subsequently cold-stored tubers of the 10 high and low BI genotypes and separated by 2D-PAGE. An exemplary gel (Figure 2) shows distribution, number and relative abundance of potato tuber proteins. On average, 790 spots could be detected when crude protein extracts were separated on a 24 cm nonlinear pH gradient ranging from 3 to 10 followed by a 12% Tris-Glycine SDS-PAGE. The most abundant protein spots were patatin isoforms (Figure 3, region R6), the main storage protein of potato tubers.47,48 Patatin isoforms clustered at a pI of approximately 4 to 5 and at a molecular weight of about 40 kilo Dalton (kDa). The lower region of the gel (
