Comparative Proteomics of Tuber Induction, Development and

Comparative Proteomics of Tuber Induction, Development and Maturation Reveal the Complexity of Tuberization Process in Potato (Solanum tuberosum L.) Lalit Agrawal, Subhra Chakraborty, Dinesh Kumar Jaiswal, Sonika Gupta, Asis Datta, and Niranjan Chakraborty* National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi-110067, India Received January 30, 2008

Tuberization in potato (Solanum tuberosum L.) is a developmental process that serves a double function, as a storage organ and as a vegetative propagation system. It is a multistep, complex process and the underlying mechanisms governing these overlapping steps are not fully understood. To understand the molecular basis of tuberization in potato, a comparative proteomic approach has been applied to monitor differentially expressed proteins at different development stages using two-dimensional gel electrophoresis (2-DE). The differentially displayed proteomes revealed 219 protein spots that change their intensities more than 2.5-fold. The LC-ES-MS/MS analyses led to the identification of 97 differentially regulated proteins that include predicted and novel tuber-specific proteins. Nonhierarchical clustering revealed coexpression patterns of functionally similar proteins. The expression of reactive oxygen species catabolizing enzymes, viz., superoxide dismutase, ascorbate peroxidase and catalase, were induced by more than 2-fold indicating their possible role during the developmental transition from stolons into tubers. We demonstrate that nearly 100 proteins, some presumably associated with tuber cell differentiation, regulate diverse functions like protein biogenesis and storage, bioenergy and metabolism, and cell defense and rescue impinge on the complexity of tuber development in potato. Keywords: tuber development • storage proteins • differential display • mass spectrometry • cluster analysis • ROS pathway

Introduction Potato is the most important noncereal food crop and ranks fourth in terms of total global food production, besides being used as animal feed, as raw material for manufacture of starch, alcohol and other food products.1 The United Nations has declared 2008 as the ‘International Year of the Potato’, affirming the need to focus on the role that the potato can play in providing food security.2 Currently, potatoes are grown in nearly 125 countries and more than a billion people worldwide consume them on a daily basis.3 While in developing countries the majority of potato is used for direct consumption, a shift toward the use of potato in convenience foods, for instance, chips and fries has been dramatically increased in developed countries.4 Potato tubers represent the underground stems that undergo a series of morphological changes involving an interaction of genetic, biochemical and environmental factors. Tuberization in potato primarily involves stolon formation, tuber induction and development, and resource storage. The major processes that have been identified during tuber life cycle are related to resource metabolism and the regulation of these processes.5 There is now abundant evidence that the highest * To whom correspondence should be addressed. Dr. Niranjan Chakraborty, National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi-110067, India. Tel: 00-91-11-26735178. Fax: 00-91-11-26741658. E-mail: [email protected]. 10.1021/pr8000755 CCC: $40.75

 2008 American Chemical Society

rate of change in gene expression occur just prior to and during tuber formation and that this activity falls off as tubers grow. Under conditions of a short-day photoperiod and low temperature, a transmissible signal is activated that initiates cell division and expansion and a change in the orientation of cell growth in the subapical region of the stolon tip. In this signal transduction pathway, perception of the appropriate environmental cues occurs in leaves and is mediated by phytochrome and gibberellins.6,7 Phytohormones also play a prominent role in regulating the morphological events of tuberization activated in the stolon tip.8 Several of the transcription factors have been reported to be involved in regulation of growth and meristem development in potato, including tuber formation by enhancing or repressing the activity of specific target genes.9–11 Further, a wide variety of nonspecific lipid transfer protein (nsLTP) genes are expressed at key stages of potato tuber life cycle.12 A recent microarray study showed the involvement of more than 1300 genes during tuber development,13 although their direct involvement in regulating many key processes remains ambiguous. It is increasingly clear that, as compared to transcripts level, the level of protein integrates post-transcriptional and post-translational processing that modulates the quantity, localization and efficiency of the final cell products. Thus, proteome analysis has become an indispensable source of information about protein expression, splice variants and Journal of Proteome Research 2008, 7, 3803–3817 3803 Published on Web 08/02/2008

research articles erroneous or incomplete prediction of gene structures in databases. This technology allows the global analyses of gene products in cells, organelles and physiological state of cells.14–16 In recent years, several reports have been published focusing changes in protein expression profile in microtuber,17 tuber life cycle,18 during storage and dormancy breaking,19,20 sinksource transition,21 besides changes in natural variants and landraces.22 Nevertheless, our knowledge on the extent of environmental influence and other physiological and developmental processes affecting tuberization are still limited. It is conceivable that there may be many other classes of proteins, as yet unidentified, which assist the tuber development process in potato. In plants, the process of development is derived from meristems and much of the action of development occurs where organs are formed in and around meristems. Organogenesis involves an early patterning stage that roughs out boundaries and facilitates the development of organs. Within these boundaries, groups of founder cells divide and growth occurs, leading to the formation of structures containing arrays of differentiated cells. Increasing evidence suggest that reactive oxygen species (ROS) play roles in cell growth and that spatial regulation of ROS production is an important factor controlling plant organs.23 We report here a systematic screening of temporal changes in protein expression during the process of tuberization in potato. The comparison of the organogenesisresponsive proteome revealed predicted and unexpected components emphasizing their possible role in tuber induction and development. Further, the results showed a temporal accumulation of enzymes that catabolize ROS, specifically during tuber initiation and enlargement, indicating the involvement of these components in tuber development. The differential expression profiles of the candidate proteins may provide new insight into the underlying mechanisms involved in tuber development. This may also facilitate the targeted alteration of metabolic routes in tubers for industrial exploitations besides generation of markers for precision selection in potato breeding program.

Experimental Section Plant Growth and Tuberization. The plants from sizenormalized seed tubers of diploid potato variety, A16 (Solanum tuberosum L.) were grown in randomized plots consisting of 30 tubers for almost 100 days, until tuber maturation. The distance among the rows in the replication plots was 60 cm and among the plants was 20 cm. The tissues were collected from different developmental stages and stored at -80 °C after quick-freezing in liquid nitrogen unless described otherwise. Isolation of Tuber Proteins. The tuber-specific soluble proteins were isolated from different development stages of tuber, pooled from three randomized plots, as described24 with few modifications. In brief, the tissues were ground to powder in liquid nitrogen and transferred to an open-mouthed 50 mL tube. Immediately, tissue powder was homogenized in homogenizing buffer [50 mM Tris-HCl (pH 8.2), 2 mM EDTA, 20% glycerol, 5 mM DTT and 2 mM PMSF]. The soluble proteins were recovered as supernatant by centrifugation at 8000g for 10 min at 4 °C. Without disturbing the pellet, the supernatant was taken in a 15 mL tube and ultracentrifuged at 117 000g for 45 min at 4 °C. The supernatant, so obtained, was the soluble protein fraction. The concentration of protein extract was determined by Bradford assay (Bio-Rad, CA). 3804

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Agrawal et al. 2-Dimensional Gel Electrophoresis. The tuber proteins were diluted in dilution buffer [100 mM, Tris-Cl (pH 8.5), 20% (v/v) glycerol, 8% (w/v) SDS, 20 mM DTT, 1 mM PMSF] and boiled for 5 min.25 Protein samples were allowed to cool to room temperature (25 °C), precipitated with 9 vol of 100% chilled acetone overnight at -20 °C. The precipitates were recovered by centrifugation at 10 000g at 4 °C, for 10 min. Protein pellets were washed twice with 80% acetone to remove excess SDS, air-dried, and resuspended in 2-D rehydration buffer [8 M urea, 2 M thiourea, 4% (w/v) CHAPS, 20 mM DTT, 0.5% (v/v) Pharmalyte (pH 4-7) and 0.05% (w/v) bromophenol blue]. Isoelectric focusing was carried out with 250 µg of protein. Protein was loaded by in-gel rehydration method onto 13 cm IEF strips (pH 4-7) and electrofocusing was performed using IPGphor system (Amersham Biosciences, Bucks, U.K.) at 20 °C for 25 000 Vh. The focused strips were subjected to reduction with 1% (w/v) DTT in 10 mL of equilibration buffer [6 M urea, 50 mM Tris-HCl (pH 8.8), 30% (v/v) glycerol and 2% (w/v) SDS], followed by alkylation with 2.5% (w/v) iodoacetamide in the same buffer. The strips were then loaded on top of 12.5% polyacrylamide gels for SDS-PAGE. The electrophoresed proteins were stained with silver stain plus kit (Bio-Rad, CA). The gel images were digitized with FluorS imaging system (Bio-Rad, CA) equipped with a 12-bit camera. Protein spot detection, quantification, and quality scoring were obtained by PDQuest software, version 7.2.0 (Bio-Rad, CA). Spot quality is a numerical value ranging from 0-100 that is calculated based on several attributes. Each attribute is evaluated and weighed to produce the numerical value. If a protein-spot fits the Gaussian model perfectly, has no streaking in X or Y direction, does not overlap with any other protein spot and has peak intensity within the linear range of scanner, it is assigned with a quality score of 100. Experimental molecular mass and pI were calculated from the digitized images using standard molecular mass marker proteins. The “low-quality” spots were assigned to the protein spots with a quality score less than 30 and were eliminated from further analysis. The remaining high-quality spot quantities were used to calculate the mean value for a given spot, and this value was used as the spot quantity on the standard gel. MS/MS Identification of Proteins. Protein spots were excised mechanically using pipet tips, in-gel digested with trypsin and peptides extracted according to standard techniques.15,16 Peptides were analyzed by electrospray ionization time-of-flight mass spectrometry (LC/MS/TOF) using an Agilent 1100 Series HPLC system (Agilent Technologies) coupled with a Q-STAR Pulsar imass spectrometer (Applied Biosystems). Tryptic peptides were loaded onto a Zorbax SB-C18 column (15 cm length; Agilent Technologies) and separated with a linear gradient of water/acetonitrile/0.1% formic acid (v/v). The MS/MS data was extracted using Analyst Software v.1.4.1 (Applied Biosystems). Peptides were identified by searching the peak-list against the MSDB 20060831 (3 239 079 sequences; 1 079 594 700 residues) database using the MASCOT v.2.1 (www.matrixscience.com) search engine. Since the potato genome sequence is not known, a homology based search was performed. The database search parameters were taxonomy, Viridiplantae (Green Plants; 247 889 sequences); peptide tolerance, (1.2 Da; fragment mass tolerance, (0.6 Da; maximum allowed missed cleavage, 1; instrument type, ESI-QUAD-TOF. Protein scores were derived from ions scores as a nonprobabilistic basis for ranking protein hits and the protein scores as the sum of a series of peptide scores. The score threshold to achieve p < 0.05 is set by Mascot

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algorithm, and is based on the size of the database used in the search. The details regarding the precursor ion mass, expected and theoretical molecular weight, delta, score, rank, charge, number of missed cleavages, p-value and the peptide sequence for proteins identified with a single peptide are mentioned in Supplemental document 1. Further, the fragment spectra for these proteins are provided in Supplemental document 2. The SOTA (self organizing tree algorithm) clustering was performed on the log transformed fold induction expression values across four stages of potato tuberization by using Multi Experiment Viewer (MEV) software (TIGR). The clustering was done with Pearson correlation as distance with 10 cycles and maximum cell diversity of 0.8.26 This algorithm is a neural network that grows adopting the topology of a binary tree, and the result of the algorithm is a hierarchical cluster obtained with the accuracy and robustness of a neural network.27 The SOTA tree summarizes the expression patterns of all proteins. Each branch represents the centroid expression profile of a group of proteins. Immunoblot Analysis. Proteins for SDS-PAGE were extracted from four different stages of tuber as described above. Onehundred micrograms of protein from each stage was subjected to SDS-PAGE on 12.5% (w/v) acrylamide Laemmli gels (7 cm). The electrophoresis was performed at room temperature and the proteins were electroblotted to nitrocellulose membrane (Amersham Biosciences, Bucks, U.K.) at 150 mA for 2 h. The membrane was blocked with 5% (w/v) nonfat milk in TTBS buffer (0.1 M Tris, pH 7.9, 0.15 M NaCl, 0.1% Tween 20). The resolved proteins were probed with the primary polyclonal antibody (Abcam Ltd., U.K. or Santa Cruz Biotechnology, Inc., CA) diluted to varying ratios (1:250-1:1000) in Tris-buffered saline (TBS). Immunodetection was performed by incubation of membrane-bound proteins with alkaline phosphatase conjugated anti goat/sheep IgG as secondary antibody. Activity Assay of ROS-Catabolizing Enzymes. Tuber tissues (250 mg) were ground in liquid nitrogen to fine powder and homogenized in 3.0 mL of KPO4-buffer (pH 7.0) for ascorbate peroxidase (APx) and 100 mM Triethanolamine (Tea, pH 7.4) for superoxide dismutase (SOD), respectively. The homogenate was centrifuged at 16 000g for 20 min at 4 °C. The supernatant was transferred into fresh tube and was used for the assay of SOD, APx and catalase.

Figure 1. Representative morphology of various stages of tuberization: stolon, initial tuber, developing tuber, and mature tuber. The four developmental stages sampled (A) were based on the tuber weight and diameter as detailed in Experimental Section. The fresh weight (B) and the diameter (C) of the harvested tubers were determined and plotted against respective stages of development. Data represent means ( SD of three measurements.

SOD activity was determined by spectrophotometric method based on the inhibition of superoxide-driven NADH oxidation.28 The assay mixture contained 100 mM Triethanolamine (Tea, pH 7.4), 100 mM/50 mM EDTA/MnCl2, 7.5 mM NADH and 10 mM mercaptoethanol in a total volume of 1.0 mL. The oxidation of NADH was followed at 340 nM (an absorbance coefficient of 6.2 mM-1 cm-1). The oxidation rates were initially low, then increased progressively (usually 2-4 min after mercaptoethanol addition) to yield linear kinetics (12-15 min), which were used for calculation. APx was assayed from the decrease in absorbance at 290 nM (an absorbance coefficient of 2.8 mM-1 cm-1) as ascorbate is oxidized by its activity.29 The reaction mixture for the peroxidase contained 50 mM potassium phosphate (pH 7.0), 0.5 mM ascorbate, and 0.1 mM H2O2 in a total volume of 1.0 mL. The reaction was initiated by adding H2O2, and the absorbance was recorded 30 s after the addition. Correction was done for the low, nonenzymatic oxidation of ascorbate by H2O2. The catalase assay was performed using 50 µg of protein for each reaction and the reaction mixture was prepared by adding 50 µL of protein extract to 925 µL of 70 mM potassium phosphate buffer (pH

7.5).16 Reaction was started by addition of 25 µL of H2O2 (3% v/v) and the decrease in absorbance at 240 nm was followed for 5 min. Baseline correction was done by subtracting the absorbance taken without addition of H2O2. The assay was done in triplicates and the enzyme activities were plotted against time. RNA Gel-Blot Analysis. RNA gel-blot analysis was done as described earlier.30 Total RNA was extracted from four different stages of tuberization in potato with TriPure isolation reagent (Roche Diagnostics). With the use of formaldehyde as a denaturant, 20 µg of total RNA was subjected to gel electrophoresis. Ethidium bromide staining under UV light was used to ascertain equal gel loading and efficient transfer to nylon membrane. The candidate genes of SOD, APx, MDHAR and MAPK were PCR amplified by the respective gene-specific primers (SODF, 5′-CAAGCAAATTGACGGAACAG-3′; SODR, 5′TCATCCTTTCGGTTTTCTCG-3′; APxF, 5′-AGCCCATTAGGGAGCAGTTT-3′; APxR, 5′-TTCCCGTCACCATTTTAAGC-3′; MDHARF, 5′-CGGAGAAGATTTCACAAACCA-3′; MDHARR, 5′-CCAGTGAGJournal of Proteome Research • Vol. 7, No. 9, 2008 3805

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Figure 3. Stage-dependent changes of few of the differentially expressed proteins during tuberization. The boxed areas (A-E) are zoomed-in gel sections and correspond to the marked areas in Figure 2B.

kinases. Quantitation of the relative kinase activities was done using a PhosphorImager (Molecular Dynamics, Inc., CA).

Results and Discussion

Figure 2. 2-DE analysis of the potato proteome during tuberization. (A) Proteins were extracted from different stages of tuberization and equal amounts (250 µg) of proteins were separated by 2-DE as described in Experimental Section. Three replicate silver-stained gels for each stage were computationally combined using PDQuest software and four representative standard gel images were generated. (B) A higher level matchset was created in silico from these standard gels. The boxed areas marked with dotted lines represent the zoomed-in gel sections in Figure 3. The numbers correspond with the spot IDs listed in Table 1.

CAAGGGAAGTCT-3′); MAPKF, 5′-GGGCTAGCTCGTGTCACTTC3′; MAPKR, 5′-ATGCGCTGGGTATTCAGGATT-3′ and used as probes. [R-32P]dCTP was incorporated into respective probes according to the manufacturer’s instructions (Perkin-Elmer). The membranes were hybridized in 50% formamide (w/v) hybridization buffer at 42 °C for 18 h. Washing was as follows: 2× SSC at room temperature for 5 min, 2× SSC and 0.1% (w/v) SDS at room temperature for 5 min, 0.1× SSC and 0.5% (w/v) SDS at 42 °C for 20-30 min. The films were exposed to Kodak X-ray film and autoradiographed. In-Gel Kinase Assay. The in-gel kinase assay was performed as described previously.31 Briefly, tuber extracts containing 20 µg of protein were separated in a 12.5% SDS-PAGE copolymerized with myelin basic protein (MBP, 500 µg mL-1), a known MAPK substrate. After denaturation and renaturation of the gel, protein kinase activity against MBP was detected by incubating the gel with γ-32P-ATP (50 µCi), followed by removal of the unincorporated label and autoradiography. Prestained size markers (Bio-Rad, CA) were used to calculate the size of 3806


Monitoring Changes in Protein Expression by 2-DE. The entire process of tuber development is considered to be divided in three general categories: (1) tissue differentiation and the development of tuber primordium; (2) tuber initiation characterized by swelling of the stolon tips; and (3) tuber development and maturation.32 Thus, stage-specific tissues were harvested from stolons at 6-8 weeks (S1, stage-1), swollen stolons or initial tubers at 8-10 weeks (S2, stage-2), developing tubers at 10-12 weeks (S3, stage-3) and mature tubers at 14-15 weeks (S4, stage-4) as shown in Figure 1A. In order to maintain uniformity, a particular range of weight and diameter of the developing tubers were considered and used for downstream analysis. The average weights of each of the development stages (stolon, initial tuber, developing tuber, and mature tuber) were 75, 510, 935 and 2165 mg, and that of diameter were 3.125, 5.75, 9.125 and 14.00 mm, respectively (Figure 1B,C). The changes in the tuber proteome were monitored for four different stages of tuberization using 2-DE analysis. For each stage, the samples were collected from three randomized plots and pooled to normalize the effect of variations in the biological replicates, if any. Three replicate 2-DE gels were run that were then computationally combined into a representative standard gel, the first level matchset (Figure 2A). The replicates had a correlation coefficient of variation above 0.8 as displayed in the scatter plots (Supplemental Figure 1). Further, the gels showed more than 90% high quality protein spots suggesting high reproducibility among the replicates (Supplemental Table 1). Protein spot detection and quantification were obtained using normalized spot volumes given by PDQuest software using the total spot volume normalization procedure to discard experimental variations in 2-DE gels. Each spot included on the standard gel met several criteria: it was present in, at least, two of the three gels and was qualitatively consistent in size and shape in the replicate gels. A second level matchset was

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Developmental Changes of Potato Proteins during Tuberization Table 1. Catalogue of Differentially Expressed Potato Proteins Associated with Tuberization

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a Spot no. as given on the 2-D gel image (Figure 2B). The spot no. was defined as StC, where St identifies the organism (Solanum tuberosum) and C identifies cytoplasm and the numeral indicates the spot nos. b Gene identification number as in GenBank. c The expression data in terms of fold-induction was log transformed to the base two and these values were plotted against stages.

then developed (Figure 2B), which allowed comparison of the standard gels from each of the stage and a second normalization was done with a set of three unaltered spots identified from across the stages. The filtered spot quantities from the standard gels were assembled into a data matrix of high-quality spots from all the representative gels for further analysis. Quantitative image analysis revealed around 219 protein spots that changed their intensities by more than 2.5-fold at least at one developmental stage. Out of these, 74 proteins were distinctly up-regulated, 67 proteins were down-regulated, while the rest showed a mixed pattern of stage-dependent expression. A total of 97 differentially expressed protein spots were identified by ion trap LC-MS/MS, which are indicated on the higher level matchset (Figure 2B). The expression profile for these protein spots is provided as Supplemental document 3. While most of the protein spots showed quantitative changes, some spots also showed qualitative change. Five typical gel regions representing protein spots with altered expression are enlarged and shown in Figure 3. Functional Classification of Proteins. To understand the function of the proteins associated with the process of tuberization, the differentially expressed proteins were sorted into five categories (Figure 4A and Table 1). Within the larger goal of global protein expression profiling during tuberization, we aimed to identify the metabolic pathways operating in the process. The differentially expressed proteins appeared to be involved in different pathways, viz., glycolysis, sucrose and starch synthesis, and defense and rescue. The largest percentage of the identified proteins was involved in biogenesis and storage (29%), bioenergy and metabolism (21%), and cell defense and rescue (12%) (Figure 4A). In a number of cases, many proteins were represented by multiple isoelectric forms (Table 1), suggesting the possible post-translational modification of the candidate protein. It is expected that, as genome resources of potato improve, the high-resolution 2-DE maps can be used as a predictive tool to search for unexpected isoelectric species to unravel possible posttranslational regula-

tion. Although adept at resolving isoelectric species, the technique of 2-DE is somewhat restricted at quantifying lowabundance proteins. For instance, as revealed by genomic information, many candidates implicated in the process of tuberization such as GA 20oxidase1, StBEL, POTH1, PHOR1, POTM1 and StCDPK could not be detected, presumably due to their low abundance. Protein Associated with Bioenergy and Metabolism. In developing tubers, glycolysis is an important platform for carbon assimilation wherein UDP-glucopyrophosphorylase (UGPase) catalyzes the reversible production of Glc-1-P from UDP-Glc. In general, the UGPase (StC-174, 175 and 218) showed induced expression pattern, excepting protein spot StC120 that showed gradual decrease (Table 1). The high induction of UGPase can be attributed to increased synthesis of sucrose during tuber development and its crucial role in glycolysis, as it corroborates the composite expression profiles of glycolytic enzymes, like fructokinase (FK, StC-44, 134 and 138), fructose bis phosphate aldolase (FBA, StC-337), glyceraldehyde-3phosphate dehydrogenase (GAPDH, StC-300) and phosphoglycerate kinase (PGK, StC-181). The UGPase could also be involved in cell wall biogenesis because the product of UGPase, UDP-Glc is used in the biosynthesis of cell wall polysaccharides.33 Pyrophosphate-dependent FK catalyzes conversion of F-6-P and F-1,6-bisP and is regulated by Fru-2,6 bisphosphate.34 The FKs showed an increased expression during tuber development and maturation. However, the expression of FBA that catalyzes the aldol cleavage of Fru 1,6-bisP to glyceraldehyde 3-P (GAP) and dihydroxyacetone phosphate (DHAP) was increased during tuber maturation. The other important finding during tuber maturation was the increased expression of GAPDH. In plants, ADP-Glc serves as the substrate for starch synthesis and ADP glucose phosphorylase (AGPase, StC-251) is used in the reversible conversion of Glc-1-P to ADP-Glc. The induced expression of AGPase was observed during tuber development, which was increased further at tuber maturity, indicating the Journal of Proteome Research • Vol. 7, No. 9, 2008 3811

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Figure 4. (A) Functional classification of differentially expressed proteins during tuberization in potato. The putative functions were assigned to each of the candidates using protein function database and grouped as represented in the pie-chart. (B) Clusterogram of 97 differentially expressed proteins showing 10 clusters based on their expression profiles. The SOTA cluster tress is shown at the top and the expression profiles are shown below. The expression profile of each individual protein in a cluster is depicted by gray lines, while the mean expression profile is marked in pink for each cluster. Detailed information on proteins within each cluster can be found in Supplemental Figure 2. S1, stolon; S2, initial tuber; S3, developing tuber; S4, mature tuber.

higher accumulation of starch in mature tubers. It is important to note that malate dehydrogenase (MDH, StC-298 and 344) and NADP dehydrogenase (NDH, StC-241), the two important components of Kreb cycle, were highly induced during tuber development. The NDH is an analogous to the NADH dehydrogenase or complex I of the mitochondrial respiratory chain that mediates the electron transfer from NADH to plastoquinone. The amount of NDH polypeptides and NADH dehydrogenase activity of the NDH complex increases under photooxidative stress and is accomplished by H2O235 and H2O2stimulated phosphorylation of the Thr-181 of the NDH-F subunit.36 The prevalence of enzymes associated with carbohydrate metabolism is in good accordance with the previous findings in potato by transcriptome analysis.13 Cell Defense and Rescue. The generation of reactive oxygen species (ROS) is an inevitable process in all aerobic organisms, especially when they encounter pathological and physiological stress conditions.37 To cope with the deleterious effects of ROS, aerobic organisms express various antioxidant proteins includ3812


ing superoxide dismutase (SOD), catalase and ascorbatedependent peroxidase (APx).38 There have been reports on increased accumulation of APx and dehydroascorbate reductase during tuber development.18 We observed increased expression of APx (StC-192 and 423), monodehydroascorbate reductase (MDHAR, StC-295), dehydroascorbate reductase and glutathione reductase. It is understood that H2O2 scavenging is accomplished by catalase, various peroxidases and the ascorbate-glutathione cycle involving these enzymes.29 Further, there was a high induction in FeSODs (StC-193), which might assist in management of low temperature induced ROS in developing tubers. This management could be either directly by increasing the scavenging capacity for superoxide and/or indirectly by increasing the flux through the H2O2 pool, thereby modifying cell signaling processes.39 Recent studies have demonstrated that expression of aldehyde dehydrogenase (StC-294) is an important determinant of resistance to toxicity caused by intermediate-chain-length aldehydes that are produced during lipid peroxidation.40 Our


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Figure 5. Immunoblot analysis of MDH, GAPDH, ADH and 14-33. Each lane was loaded with 100 µg of protein, resolved on 12.5% SDS-PAGE and electrotransferred onto nitrocellulose membrane. The membranes were probed with respective primary polyclonal antibodies and the proteins were detected by incubation with alkaline phosphatase-conjugated antigoat/sheep IgG as secondary antibody. The representative Coomassie-stained gel shows uniform protein loading. MDH, malate dehydrogenase; GAPDH, glyceraldehyde 3-P dehydrogenase; ADH, alcohol dehydrogenase. S1, stolon; S2, initial tuber; S3, developing tuber; S4, mature tuber.

observation on increased activity of aldehyde dehydrogenase suggests the possible defense strategy in detoxification of aldehydes during tuber development. A positive correlation between the accumulation of late embryogenesis abundant (LEA) transcripts or proteins and cold acclimation has been suggested.41,42 We also found increased accumulation of LEA (StC-355) in phase with the cold-induced tuber development process. Protein Biogenesis and Storage. The patatin protein family represents the primary storage protein in potato tubers and is a critical nutritional component. The members of patatin family are mostly expressed in tubers and highly activated following tuber initiation.5 Increasing evidence suggest that the patatins exhibit alterations in chromatin state and differential transcriptional regulation during the developmental transition from stolons into tubers.43 In this study, many patatins (StC-39, 40, 42, 45 and 104) showed increased expression with the continued increase in tuber size, while several others (StC-154, 438 and 471) were highly expressed in stolons indicating their role in tuber initiation. The expression pattern of patatin protein family appears to be similar to what was detected by cDNA microarrays.13 Low-molecular-weight protease inhibitors are the second major storage proteins in potato tubers. Of these species, the protease inhibitors I and II44,45 and a set of Kunitz-type enzyme inhibitors46 accumulate during tuber development. We found the similar patterns of expression profiles for Kunitz-type enzyme inhibitors (StC-18, 20 and 271), aspartic protease inhibitors (StC-51 and 151), cysteine protease inhibitor (StC58) and serine protease inhibitor (StC-103). All of these enzymes showed maximum abundance in mature tubers suggesting a storage property. In contrast, several other protease inhibitors (StC-8, 379, 437, 438, 440, 470 and 447), which were abundant in early stage of tuber initiation, disappeared with the progressive development of the tuber. Protein disulfide isomerase (PDI, StC-185), a multifunctional protein,47 catalyzes the formation of disulfide bonds during protein biogenesis.48 PDI markedly increases reactivation of

Figure 6. Activities of ROS-metabolizing enzymes and their expression in stolon, initial tuber, developing tuber, and mature tuber. The ROS pathway enzymes, viz., SOD (A), catalase (B), and APx (C) were extracted and the activities were assayed. The histograms show the relative expression patterns of the enzymes. Activities of SOD, catalase and APx are expressed as nM NADP oxidized mg-1 protein min-1, nM H2O2 oxidized mg-1 protein min-1 and nM ascorbate oxidized mg-1 protein min-1, respectively. Data represent means ( SD of three assays at each stage. Northern blot analysis (D) was done with 20 µg of total RNA, extracted from each stage during tuberization. Ethidium bromidestained rRNA served as a loading control. S1, stolon; S2, initial tuber; S3, developing tuber; S4, mature tuber.

GAPDH and prevents their aggregation,49 and thus, the identical expression profile of PDI and GAPDH is indicative of their cooperative role during tuber development. Journal of Proteome Research • Vol. 7, No. 9, 2008 3813

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Figure 7. Analysis of MAPK in stolons and different stages during tuber development. Northern analysis (A) was performed with 20 µg of total RNA from each stage, resolved into 1.2% agarose gel and probed with a labeled 0.5-kb MAPK gene. Ethidium bromide-stained rRNA was used as loading control. The immunoblot analysis of MAPK (B) was performed with 100 µg of tuber protein as described in Figure 5. The Coomassie-stained gel serves as loading control. In-gel protein kinase assay (C) was carried out with tuber extracts containing 20 µg of protein per lane separated by SDS-PAGE. MBP (0.5 mg/mL) was used as substrate. S1, stolon; S2, initial tuber; S3, developing tuber; S4, mature tuber. The histogram represents the relative activities of MAPK at different stages of tuber development.

Miscellaneous Proteins. In addition to major protein classes, several important candidates like 14-3-3 (StC-13), Ran-binding protein (RanBP, StC-49), selenium binding protein (StC-228), soluble NSF-attachment protein (StC-82) and F-box protein ORE9 (StC-150) were expressed differentially during tuber development. The RanBP showed gradual increase in expression with concomitant increase in tuber size. In the dimeric complex, RanBP1 is highly specific for the GTP-bound form of Ran, which plays important roles during cell division.50 14-3-3 proteins are ubiquitous eukaryotic proteins that have wideranging regulatory functions. These proteins serve either to directly regulate the activity of interacting proteins, or to modify the intracellular localization of their targets.51 Many other hypothetical proteins (StC-23, 168, 187, 293 and 318) were found to be differentially regulated, which are subject to further studies in establishing their contributions in the process of tuberization. Dynamics of Protein Networks during Tuberization. To summarize the information presented in Table 1 and to cluster the proteins showing similar expression profiles during tuberization, hierarchical clustering was applied to the differentially expressed proteins. To achieve a comprehensive overview of expression profile in terms of protein function, the data sets were subjected to unbiased clustering method, SOTA. The analysis yielded 10 expression clusters for four different stages of tuberization (Figure 4B). Only the clusters with n g 4 were taken to study the coexpression patterns for functionally similar proteins. The detailed information on proteins within each cluster is presented in Supplemental Figure 2. The proteins involved in biogenesis and storage destination and bioenergy and metabolism mainly displayed an early induction at stolons and maintained almost steady state henceforth. This class has a large number of storage proteins, such as patatins (cluster 2 and 4). A few members of patatin protein family were found to be downregulated (cluster 10), which is in agreement with the earlier observations.43 Enzymes 3814


Agrawal et al. involved in carbon assimilation pathway were found to be differentially downregulated during tuberization. The expression profiles varied for different classes of proteins and no clear clustering was observed at any particular stage of tuber development. Notably, tuberization is a dynamic process, which seems to maintain a delicate balance between carbon assimilation and metabolism. The formation of storage proteins and starch needs a constant source of energy along with storage product formation. Cell defense proteins, which showed a late expression that is, from stage-2 onward comprises mainly HSPs and chaperonins (cluster 6 and 10). However, proteins involved in the production and scavenging of ROS were induced at early stages of tuber development (cluster 1 and 2). These results, altogether, suggest that the storage reserves are built during tuberization and proteins involved in assimilation of these products are predominantly expressed. Investigation of Selected Proteomic Data by Immunoblot Analysis. The list of putatively regulated proteins depicted in Table 1 is a snapshot of proteins from stolon to tuber induction, development, and maturation. It is estimated that 2% of the fresh weight of a potato tuber is present as protein whereas 20% is represented as starch.52 A high supply of carbohydrates to the developing stolons favors tuber induction and is a major step for tuber initiation and enlargement. In developing tubers, carbohydrate metabolism is accompanied by many overlapping signal transduction events wherein phosphorylation-induced association of 14-3-3 proteins might play a key role. Further, 14-3-3s also can bind certain nonphosphorylated targets, indicating that they also have roles outside the context of phosphorylation-mediated signal transduction. We observed highly significant differential expression of several of the carbohydrate metabolism associated enzymes, viz., MDH, GAPDH and alcohol dehydrogenase. While GAPDH (StC-300) catalyzes the conversion of GAP into 1,3-bis PGA in glycolysis, MDH (StC-298 and 344) catalyzes the conversion of malic acid to oxaloacetic acid in Kreb cycle and alcohol dehydrogenase (StC-334) converts lactic acid into alcohol. Thus, the expression profiles of these enzymes along with that of 14-3-3 family proteins were investigated during tuber development by immunoblot analysis. As shown in Figure 5, the expressions of MDH and GAPDH were significantly induced in initial tuber (stage-2) and maintained steady state during tuber development (stage-3 and 4). Similar was the accumulation pattern of ADH whose expression was low in stolon (stage-1), increased at early stage and continued until tuber maturity. The 14-3-3 family proteins began to increase expression at early tubers (stage-2), and further increased in developing tubers (stage3). Though the trend in expression profile of these proteins was somewhat similar as determined by the 2-D analysis (Table 1), there was difference in fold-induction in protein expression. Since all the differentially expressed proteins were not identified, it can be assumed that some other isoforms of the same protein might be part of the tuber proteome. Further, posttranslational modifications of some of the differentially expressed proteins might also affect the actual expression levels determined by two different techniques. Temporal Changes in Expression of ROS-Catabolizing Enzymes. In potato, stolons grow as horizontal stems in noninductive conditions and under low temperature or short day conditions, elongation growth of the stolon ceases thereby facilitating tuber formation. It is likely that the influence of low temperature may give rise to increased ROS levels, particularly superoxide radicals (O2•-) and H2O253,54 in developing tubers.


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Figure 8. Pathways involved in carbon assimilation, source-sink transport, and protein biogenesis and storage in potato during tuber development. Proteins involved in sugar breakdown, ROS pathway, and storage are displayed on the corresponding metabolic pathways. Graphs are the representatives of expression profile of individual protein and number given below in each graph indicates the protein identification number. Abbreviations for metabolites: UDP-G, UDP-Glc; ADP-G, ADP-Glc; G-1-P, Glc 1 phosphate; G-6-P, Glc 6 phosphate; F-6-P, Fru 6 phosphate; F-1,6-bis P, Fru 1,6 bis phosphate; GAP, glyceraldehyde 3-phosphate; DHAP, dihydroxyacetone phosphate; 1,3-bis PGA, 1,3 bis phosphoglyceric acid; 3-PGA, 3 phosphoglyceric acid; 2-PGA, 2 phosphoglyceric acid; PEP, phosphoenolpyruvate; M-6-P, mannose 6 phosphate; R-KGA, R-keto glutaric acid; MA, malic acid; OAA, oxalo acetic acid. Abbreviations for proteins: UGPase, UDP-Glc pyrophosphorylase; AGPase, AGP-Glc pyrophosphorylase; PFK, pyrophosphate-dependent phosphofructokinase; FBA, Fru bisphosphate aldolase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PGK, phosphoglycerate kinase; MDH, malate dehydrogenase; ADH, alcohol dehydrogenase; NdhF, NADP dehydrogenase F subunit; M6PR, mannose 6 phosphate reductase; APx, ascorbate peroxidase; MDHAR, monodehydroascorbate reductase; ALDH, aldehyde dehydrogenase; NDK, nucleoside diphosphate kinase; LEA, late embryogenesis abundant; HSPs, heat shock proteins; KTPI, Kunitz type protease inhibitor; API, aspartate protease inhibitor; CPI, cystein protease inhibitor, SPI, serine protease inhibitor.

The SOD catalyzes the dismutation of O2•- radicals to molecular oxygen and H2O2, thus, playing a key role in cell defense and rescue mechanism.55 Subsequently, decomposition of H2O2 is accomplished by catalase or through a series of

oxidoreduction reactions involving APx and glutathione peroxidase (GPx) using ascorbate or reduced glutathione, respectively. Interestingly, besides their well-known toxic effects, H2O2 may act as secondary messengers modulating various cellular Journal of Proteome Research • Vol. 7, No. 9, 2008 3815

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Agrawal et al. 62

Figure 9. Venn diagram showing the distribution of differentially expressed proteins in stage-specific and overlapping manner during tuber induction, development, and maturation. The areas shown in the diagram are not proportional to the number of proteins in the groups.

functions.56,57 Thus, we investigated the behavior of the antioxidant enzymes, SOD, APx and catalase in cellular extract during tuberization. The enzymatic assay showed that the SOD and catalase activity increased temporarily at initial tuber (stage-2) and were gradually decreased (Figure 6A,B). Similar was the case with APx activity, which also showed slight increase at this stage of tuber development (Figure 6C). It is possible that the oxidized ascorbate produced by APx is reduced to AsA by the Halliwell-Asada pathway.58 These results suggest that the activation of the ROS pathway during tuber development could result in increase in APx activity. The expression profiles of the above-mentioned enzymes were further investigated by Northern analysis; however, the results did not match with those obtained by activity assay. The transcript levels of SOD and APx remained fairly static throughout tuberization, except slight increase during tuber maturation, indicating the possible post-transcriptional regulation of these enzymes. The level of MDHAR was found to be decreased in a time-dependent manner during tuber development (Figure 6D). The decrease in MDHAR expression can be attributed to the activation of recycling system of AsA by low temperature. Mitogen-activated protein kinases (MAPKs) are believed to play critical roles in development, cell proliferation, and hormone physiology.59 Interestingly, ROS-induced activation of MAPKs is regarded as evidence that ROS act upstream of MAPKs.60,61 To investigate the activation of MAPKs, the accumulation pattern of the StMPK1 mRNA was determined in different stages of tuber development by Northern blot (Figure 7A). Further, the accumulation of MAPK proteins was investigated by immunoblot analysis (Figure 7B). There was a positive correlation between the accumulation of MAPK transcripts and proteins. A higher degree of accumulation of MAPK was observed during tuber induction and enlargement but reduced accumulation in the mature tubers (Figure 7A,B). The in-gel kinase assay showed a band corresponding to protein kinase of 46 kDa molecular mass across all four stages during tuberization, though maximal activity was observed at tuber initiation (Figure 7C). This can be attributed to the stagespecific activation of MAPKs and their assorted function during tuberization. Concluding Remarks. Tuberization in potato is a complex developmental process that requires coordinated interactions of environmental, biochemical, and genetic factors. It involves many biological processes, including carbon partitioning, signal 3816


transduction and meristem determination. Earlier, Kloosterman et al. described the expression of more than 1300 genes using cDNA microarrays, and genes previously not known to be differentially expressed during tuber development were identified.13 This study, although useful and informative, highlights the complexity in prioritizing target genes for further study in the absence of their function. Recently, a preliminary proteomic analysis has identified 109 differentially expressed proteins during tuber development including 59 spots showing highly significant differences and remaining 50 with smaller changes in abundance.18 The proteomics approach has provided an overview of the expression of potato proteins associated with tuber life-cycle though it was compromised by limitations in the amount of materials for early developmental stages. Moreover, a coherent sequence of events involved in carbon assimilation, and protein biogenesis and storage during tuberization in potato is lacking. We found proteins that were not previously associated with tuber development as well as proteins that are reported to be involved. This study revealed many proteins, for example, phosphoglycerate kinase, UGPase, fructokinase, Kunitz-type enzyme inhibitors and patatin family proteins represented by multiple isoforms, which is consistent with the transcriptome data.13 Our observation thus confirm and extend findings regarding the potential protein components, which may be informative in elucidating how the target proteins may be modulated in different stages of tuber development (Figure 8). This would offer the basis to analyze expression profiles of many proteins within the same metabolic pathway and therefore provide additional information on metabolic flux as well as the key cellular response during tuberization. In conclusion, the tuber-specific comparative proteomes of potato identified a number of proteins that are putatively associated with tuber initiation, development and maturation. These data are particularly important, at least in part, due to the fact that despite the existence of nearly 225 000 ESTs, the currently available potato sequence data show a severe underrepresentation of proteins. Of the 97 differentially expressed proteins, 7 were exclusively expressed in the stolons, implying their possible role in stolon formation. Among the remaining, 9 proteins were found to be expressed throughout the entire process and 49 were expressed during the later stages of tuber development, highlighting their regulatory and storage functions (Figure 9). The proteins involved in biogenesis and storage constituted the most abundant class followed by those involved in bioenergy and metabolism (Figure 4A). The preponderance of metabolic proteins offers a unique opportunity to map activities for carbon assimilation and storage products like sucrose and starch. The immunoblot analysis for short-listed proteins in carbohydrate metabolism depicts an increase in starch synthesis from tuber initiation to maturation (Figure 5). In actively developing tubers, the activation of AGPase reflects the stimulation of starch synthesis and decreased levels of glycolytic intermediates possibly by linking starch synthesis to sucrose supply.63 The protein candidates presumably associated with cell defense against ROS such as SOD, APx, MDHAR, ALDH, LEA proteins, and many protease inhibitors were found to be upregulated during tuber initiation. Emerging evidence indicates that ROS, especially O2•- and H2O2, are important cell signaling molecules64 that may activate multiple intracellular proteins and enzymes, including MAPKs. Their production is regulated by hormone-sensitive enzymes such as NAD(P)H oxidases,65 and their metabolism is coordinated by antioxidant

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Developmental Changes of Potato Proteins during Tuberization enzymes. It is likely that an orchestrated balance between the generation and scavenging of ROS may play a significant role in tuber development in potato, although their exact contribution remains to be investigated.

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Comparative Proteomics of Tuber Induction, Development and

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