**3. Results and discussion**

#### **3.1. Map of patatin isoforms based on 2-DE**

Patatin isoforms in mature potato tuber of cv. Kennebec were first recognized on our 2-DE gels according to the previously reported studies on 2-DE patatin profiles [23–25]. We found that patatin profiles were constituted by a complex constellation of different spots showing large variations in *M*<sup>r</sup> and/or p*I* (**Figure 1**). Specifically, 2-DE resolved a total of 20 spots distributed Identification and Mapping of Phosphorylated Isoforms of the Major Storage Protein of Potato... http://dx.doi.org/10.5772/intechopen.70400 71

 precursors ions with a relative resolution of 300 full width at half maximum (FWHM) and metastable suppression. The 4000 Series Explorer Software v. 3.5 (Applied Biosystems) was used for mass data analysis. Combined peptide mass fingerprinting (PMF) and MS/MS fragment-ion spectra were interpreted with GPS Explorer Software v. 3.6 using Mascot software v. 2.1 (Matrix Science, Boston, MA, USA) to search against the *S. tuberosum* UniProtKB/ Swiss-Prot databases. Mascot database search parameters were: precursor mass tolerance of 50 ppm, MS/MS fragment tolerance of 0.6 Da, one missed cleavage allowed, carbamidomethyl cysteine (CAM) as fixed modification and oxidized methionine as variable modification. Identification of patatin phosphopeptides was also performed from spectrum data allowing phosphor-serine (PhosphoS), phosphor-tyrosine (PhosphoY) and phosphor-threonine (PhosphoT) residues as variable modification to search against the UniProtKB/Swiss-Prot databases. Analysis of phosphorylation sites was implemented using the Plant Protein

for validation. Proteins with at least two matched peptides and statistically significant (*p*-value

The phosphorylation rate at each spot was quantified using the measure *PR* [10]. It is defined as *PR* = [(*T* − *D*)/*T*] × 100, where *T* and *D* are the volumes of a given spot on 2-DE gels untreated (total protein volume) and treated (dephosphorylated protein volume) with HF-P, respectively. Non-parametric bootstrap confidence intervals (CIs) were obtained for mean values of *PR* across four biological replicates by the bias-corrected percentile method [34]. For each observed mean of *PR*, 2000 bootstrap samples of size N = 4 were drawn with replacement by applying a Monte Carlo algorithm. The 95 and 99% CIs for the observed mean of *PR* were constructed from distribution of 2000 bootstrap mean replications. The bootstrap estimate of bias was obtained from the proportion of bootstrap mean replications lower than the original estimate of the mean, and bias-corrected CIs were then calculated using the theoretical normal distribution as described by Efron [34]. *PR* data were clustered by using the unweighted pair-group method with arithmetic averaging (UPGMA). The UPGMA dendrogram derived from the matrix of pairwise *PR*-values was generated using NTSYSpc v. 2.1 software (Applied Biostatistics, Setauket, NY, USA). Descriptive statistics and Spearman's correlation test were calculated with the IBM SPSS Statistics 20 (SPSS, Chicago, IL, USA) statistical software

Patatin isoforms in mature potato tuber of cv. Kennebec were first recognized on our 2-DE gels according to the previously reported studies on 2-DE patatin profiles [23–25]. We found that patatin profiles were constituted by a complex constellation of different spots showing large

and/or p*I* (**Figure 1**). Specifically, 2-DE resolved a total of 20 spots distributed

< 0.05) MASCOT scores were selected as positively identified.

DB) [33]. All identifications and spectra were manually checked

Phosphorylation DataBase (P3

**2.11. Data analysis**

70 Advances in Seed Biology

package.

variations in *M*<sup>r</sup>

**3. Results and discussion**

**3.1. Map of patatin isoforms based on 2-DE**

**Figure 1.** High-resolution 2-DE reference map of the patatin isoforms in mature tuber of cv. Kennebec. The enlarged gel image shows patatin spots consecutively numbered in the order of the lower to the higher p*I*. 2-DE was performed using a 24-cm long IPG strip of linear pH 4–7 gradient in the first dimension and SDS-PAGE (10% by mass) in the second. The protein loading was 75 μg and the gel was stained with SYPRO Ruby fluorescent stain. The arrows indicate ovalbumin (45.0 kDa) marker position on the gel. The *M*<sup>r</sup> of spots was assessed from ovalbumin and standard molecular mass markers ranging from 15 to 200 kDa and their p*I*s from strips of linear pH.

into three main levels with *M*<sup>r</sup> between 40.1 and 43.0 kDa and p*I* range varying from 4.8 to 5.3. A total of 20 spots were excised from gel and identified by MALDI-TOF and MALDI-TOF/ TOF MS. The identification results are listed in **Table 1**. All but one small and weakly stained spot (spot 3) were confidently identified. MS analyses confirmed that, indeed, those spots contained only patatin polypeptides. However, most of the identifications were ambiguous and compatible with the occurrence of different types of patatin. This uncertainty is a consequence of the well-known high degree of sequence homology (at least 90%) among isoforms [19, 20]. Only protein spots with higher *M*<sup>r</sup> (spots 1, 2 and 4) were unambiguously identified as Patatin-3-Kuras 1 (PT3K1). It can be understood by considering that the pt3k1 gene exhibits the most differentiated sequence from other patatin genes according to the phylogenetic tree inferred from cDNA sequence analysis [24].

The two-dimensional map of the patatin was implemented with the location of glycosylated isoforms using the enzyme PNGase F. It is an effective enzymatic method for removing almost all *N*-linked oligosaccharides (glycans) from glycoproteins through the hydrolysis of the glycosamide linkage between the terminal GlcNAc and the Asn amide nitrogen [35]. We found that the three main spot levels in *M*<sup>r</sup> on 2-DE gels obtained from untreated samples merged to a single spot level after incubation with PNGase F, with an apparent decrease in *M*<sup>r</sup> (not shown). It indicates that variable degrees of glycosylation are a major contributor to the *M*<sup>r</sup> heterogeneity detected on 2-DE gels. High *M*<sup>r</sup> difference among patatin isoforms has been explained by the presence of up to three potential N-glycosylation sites at Asn residues [22, 24, 25, 36]. The mapping of glycosylated isoforms on 2-DE gels can be useful in future studies investigating the functional role of this post-translational protein modification (PTM) of the patatin.


a Gel position of assigned spots is shown in **Figure 1**.

b Experimental p*I* value.

c Matched peptides and percentage of the polypeptide sequence covered by matched peptides. <sup>d</sup>PT3K1, abbreviation for Patatin-3-Kuras 1.

**Table 1.** Protein, phosphopeptides and phosphosites along 2-DE patatin spots of cv. Kennebec, identified from MALDI-TOF and MALDI-TOF/TOF MS data.

#### **3.2. In-gel identification of phosphorylated patatin isoforms**

Pro-Q DPS was used for in-gel multiplex identification of phosphorylated patatin isoforms. Representative 2-DE images of patatin in mature tuber of cv. Kennebec on the same gel stained with Pro-Q DPS and post-stained with SYPRO Ruby are shown in **Figure 2**. The PeppermintStick markers used as positive and negative controls of protein phosphorylation validated the specificity of the recognition of phosphoproteins by Pro-Q DPS under our experimental conditions. It was found that all 20 patatin spots of the reference pattern exhibited Pro-Q DPS fluorescent signal. Similar result was obtained for patatins from mature tubers of cvs. Agria, Amanda and Ivory Russet (not shown). We can conclude, therefore, that phosphorylation is a ubiquitous PTM associated with isoforms of the patatin.

**Figure 2.** Mapping of phosphorylated patatin spots in mature tubers (cv. Kennebec) on 2-DE gel. (a) Reference profile of patatin spots on gel stained with the non-specific-protein SYPRO Ruby stain. (b) Profile of phosphorylated patatin spots from the same gel stained with the specific-phosphoprotein Pro-Q DPS fluorescent dye. The phosphoprotein ovalbumin was used as a marker of the reliability of Pro-Q DPS under our experimental conditions.

**3.2. In-gel identification of phosphorylated patatin isoforms**

Matched peptides and percentage of the polypeptide sequence covered by matched peptides.

Gel position of assigned spots is shown in **Figure 1**.

<sup>d</sup>PT3K1, abbreviation for Patatin-3-Kuras 1.

TOF and MALDI-TOF/TOF MS data.

a

b

c

Experimental p*I* value.

72 Advances in Seed Biology

Pro-Q DPS was used for in-gel multiplex identification of phosphorylated patatin isoforms. Representative 2-DE images of patatin in mature tuber of cv. Kennebec on the same gel stained with Pro-Q DPS and post-stained with SYPRO Ruby are shown in **Figure 2**. The PeppermintStick markers used as positive and negative controls of protein phosphorylation validated the specificity of the recognition of phosphoproteins by Pro-Q DPS under our experimental conditions. It was found that all 20 patatin spots of the reference pattern exhibited Pro-Q DPS fluorescent signal. Similar result was obtained for patatins from mature tubers of cvs. Agria, Amanda and Ivory Russet (not shown). We can conclude, therefore, that

**Table 1.** Protein, phosphopeptides and phosphosites along 2-DE patatin spots of cv. Kennebec, identified from MALDI-

**Spot no.a Exp. p***I* **<sup>b</sup> Match./cov. (%)c Mascot score Protein name (type)d No. phosphopeptides/**

 4.84 7/24 191 Patatin (PT3K1) 1/1 4.88 13/58 571 Patatin (PT3K1) 7/17 4.90 – – Unidentified – 4.93 5/16 158 Patatin (PT3K1) – 4.96 3/8 106 Patatin (various) – 4.96 5/21 >60–187 Patatin (various) – 5.02 11/40 >60–200 Patatin (various) – 5.02 6/25 >60–297 Patatin (various) – 5.05 7/28 >60–324 Patatin (various) 1/1 5.08 5/11 >60–199 Patatin (various) 1/1 5.12 9/32 >60–337 Patatin (various) 3/7 5.13 8/30 >60–298 Patatin (various) – 5.14 9/31 >60–475 Patatin (various) 1/1 5.16 9/31 >60–267 Patatin (various) – 5.20 2/11 >60–103 Patatin (various) 4/9 5.20 10/40 >60–440 Patatin (various) 4/10 5.23 5/25 >60–123 Patatin (various) – 5.25 9/31 >60–259 Patatin (various) 5/9 5.29 3/9 >60–129 Patatin (various) – 5.27 10/41 >60–550 Patatin (various) 4/8

**phosphosites**

phosphorylation is a ubiquitous PTM associated with isoforms of the patatin.

A prospective identification of phosphopeptides and phosphosites by MASCOT search using spectra data from MALDI-TOF and MALDI-TOF/TOF MS revealed 22 non-redundant patatin phosphopeptides containing 49 non-redundant phosphorylation sites (**Table 1**). Comparison with large-scale phosphoproteomic screens in other species using the Plant Protein Phosphorylation DataBase (P3 DB) [33] suggests that most phosphorylation sites identified are novel to this study. Thus, no phosphorylated ortholog sites were identified in other plant phosphoproteomics data for *Arabidopsis thaliana*, *Brassica napus*, *Glycine max*, *Medicago truncatula*, *Oryza sativa* and *Zea mays*. It is noteworthy that enrichment methods of underrepresented phosphorylated proteins or peptides can be conducted prior to highresolution MS analysis to precisely identify and map phosphorylation sites, but the amount of protein collected in a spot is often insufficient for downstream enrichment methods [37]. Consequently, it would be difficult to assign phosphorylation sites to the specific isoforms found along 2-DE patatin patterns. At the present time, the 2-DE map of phosphorylated isoforms appears to be more informative than the exact identification of phosphosites in order to evaluate their biological meaning. Regardless of this, phosphorylation site prediction analysis is an additional evidence for phosphorylation of patatin.

### **3.3. Quantitative profiling of phosphorylated patatin isoforms**

Changes in the phosphorylation level across patatin spots of cv. Kennebec were assessed by chemical dephosphorylation of total tuber protein extracts with HF-P coupled to 2-DE. This experimental approach provides more efficient information than Pro-Q DPS to the identification and quantification of phosphorylated proteins on 2-DE gels [10]. The reason is that the Pro-Q DPS fluorescent signal of spots containing low-abundance phosphopeptides is seriously suppressed by abundant non-phosphorylated phosphopeptides. The chemical dephosphorylation method has the advantage of using SYPRO Ruby stain, which combines good sensitivity with excellent linearity [38].

Representative 2-DE gel images of the patatin pattern before and after HP-F treatment are shown in **Figure 3**. First of all, note that spots of the protein phosphorylation marker, ovalbumin, underwent a basic shift on p*I* after HF-P treatment. This indicates that HF-P had sufficient time to achieve a complete dephosphorylation of polypeptides. With respect to the 2-DE profiles of dephosphorylated patatin, we can highlight two important observations. First, all spots observed in untreated samples were also present after dephosphorylation, but with an apparent decrease in volume. This suggests that patatin spots contained a mixture of phosphorylated and unphosphorylated isoforms. Accordingly, other factors together with protein phosphorylation must be contributing to charge heterogeneity along 2-DE patatin patterns such as difference in charged amino acids over isoforms [22]. Second, newly arisen spots (spots 21–27) found in dephosphorylated patatin patterns appeared on more basic positions of 2-DE gels. MS analysis confirmed that these new spots contained patatin (data not shown), and thereby they are isoforms that underwent a basic shift on p*I* after dephosphorylation with HF-P.

The phosphorylation level of each spot was evaluated with the measure *PR* using volumes obtained by PDQuest software from phosphorylated and dephosphorylated profiles. Mean (±SE, standard error) values of *PR* for each spot together with bias-corrected 95 and 99% bootstrap CIs are shown in **Table 2**. Interestingly, we found that spots were not uniformly phosphorylated: mean *PR*-values across spots were in the range of 4.6–52.3% and averaged (±SE) 34.4 ± 2.8%. The bootstrapped 95 and 99% CIs revealed statistically significant differences (p-value < 0.05) between many pairs of spots. Patatin spots were subsequently grouped into clusters from *PR*-values using a dendrogram UPGMA. The resulting dendrogram showed that spots cluster in three main groups with statistically significant mean differences in *PR* (*p* < 0.01) assessed by bias-corrected 99% bootstrap CIs (**Figure 4**). In particular, spots of the group 3 (spots 13 and 20) formed a well-separated cluster (mean *PR* = 8.3%) from the two remaining groups (the mean *PR* of groups 1 and 2 was 44.0 and 30.1%, respectively).

Consequently, it would be difficult to assign phosphorylation sites to the specific isoforms found along 2-DE patatin patterns. At the present time, the 2-DE map of phosphorylated isoforms appears to be more informative than the exact identification of phosphosites in order to evaluate their biological meaning. Regardless of this, phosphorylation site prediction anal-

Changes in the phosphorylation level across patatin spots of cv. Kennebec were assessed by chemical dephosphorylation of total tuber protein extracts with HF-P coupled to 2-DE. This experimental approach provides more efficient information than Pro-Q DPS to the identification and quantification of phosphorylated proteins on 2-DE gels [10]. The reason is that the Pro-Q DPS fluorescent signal of spots containing low-abundance phosphopeptides is seriously suppressed by abundant non-phosphorylated phosphopeptides. The chemical dephosphorylation method has the advantage of using SYPRO Ruby stain, which combines good

Representative 2-DE gel images of the patatin pattern before and after HP-F treatment are shown in **Figure 3**. First of all, note that spots of the protein phosphorylation marker, ovalbumin, underwent a basic shift on p*I* after HF-P treatment. This indicates that HF-P had sufficient time to achieve a complete dephosphorylation of polypeptides. With respect to the 2-DE profiles of dephosphorylated patatin, we can highlight two important observations. First, all spots observed in untreated samples were also present after dephosphorylation, but with an apparent decrease in volume. This suggests that patatin spots contained a mixture of phosphorylated and unphosphorylated isoforms. Accordingly, other factors together with protein phosphorylation must be contributing to charge heterogeneity along 2-DE patatin patterns such as difference in charged amino acids over isoforms [22]. Second, newly arisen spots (spots 21–27) found in dephosphorylated patatin patterns appeared on more basic positions of 2-DE gels. MS analysis confirmed that these new spots contained patatin (data not shown), and thereby they are isoforms that underwent a basic shift on p*I*

The phosphorylation level of each spot was evaluated with the measure *PR* using volumes obtained by PDQuest software from phosphorylated and dephosphorylated profiles. Mean (±SE, standard error) values of *PR* for each spot together with bias-corrected 95 and 99% bootstrap CIs are shown in **Table 2**. Interestingly, we found that spots were not uniformly phosphorylated: mean *PR*-values across spots were in the range of 4.6–52.3% and averaged (±SE) 34.4 ± 2.8%. The bootstrapped 95 and 99% CIs revealed statistically significant differences (p-value < 0.05) between many pairs of spots. Patatin spots were subsequently grouped into clusters from *PR*-values using a dendrogram UPGMA. The resulting dendrogram showed that spots cluster in three main groups with statistically significant mean differences in *PR* (*p* < 0.01) assessed by bias-corrected 99% bootstrap CIs (**Figure 4**). In particular, spots of the group 3 (spots 13 and 20) formed a well-separated cluster (mean *PR* = 8.3%) from the two remaining groups (the mean *PR* of groups 1 and 2 was 44.0

ysis is an additional evidence for phosphorylation of patatin.

sensitivity with excellent linearity [38].

74 Advances in Seed Biology

after dephosphorylation with HF-P.

and 30.1%, respectively).

**3.3. Quantitative profiling of phosphorylated patatin isoforms**

**Figure 3.** 2-DE profile of dephosphorylated patatin with HF-P in mature tuber (cv. Kennebec). (a) Reference profile of patatin without dephosphorylation treatment on gel stained with SYPRO Ruby stain. (b) Profile of patatin after chemical dephosphorylation on gel stained with SYPRO Ruby. Closed circles represent newly arisen spots on gel after dephosphorylation as compared to the reference profile. Identification of new spots as patatin was performed by MALDI-TOF and MALDI-TOF/TOF MS.

Elucidating whether changes in abundance of protein phosphorylation reflect either changes in phosphorylation status or changes in the abundance of the protein itself is a major challenge in the interpretation of quantitative phosphoproteomics studies [39, 40]. Thus, phosphopeptide enrichment methods prior to high-resolution MS permit the identification of low-abundance phosphoproteins but prevent joint quantitation of phosphorylation status and abundance of proteins [39]. However, our experimental approach allowed us to successfully tackle this problem. Thus, we have detected a statistically significant negative relationship between patatin spot volumes and their corresponding *PR*-values by Spearman's non-parametric correlation test (*rs* = −0.42, *p* < 0.001, n = 70). In addition, *PR*-values were negatively correlated with spot p*I*s (*rs* = −0.31, *p* < 0.01, n = 70). As expected under a differential phosphorylation


Volume of spots for untreated and dephosphorylated protein samples with HF-P were assessed by PDQuest software. a Gel position of assigned spots is shown in **Figure 1**.

b The bootstrap distribution is median biased if the probability (P) of ( *θ* ∧ *<sup>B</sup>* ≤ *θ* ∧ ) ≠ 0.50, which was calculated from 2000 bootstrap replicates; *θ* ∧ *<sup>B</sup>* and *θ* ∧ are the bootstrap mean and the sample mean estimates, respectively.

c CI—Confidence interval; CL—lower bound; CU—upper bound.

<sup>d</sup>N/A = not available, weakly stained spot with a volume below the limit of detection.

**Table 2.** Mean (±SE) values of *PR* for patatin spots estimated from four replicates from dormant tubers of cv. Kennebec.

hypothesis, isoforms located on acidic gel positions tended to be more highly phosphorylated than those of basic positions. It is also noteworthy that phosphorylation levels were estimated using the measure *PR*, which takes into account the amount of protein at each spot. Therefore, differential phosphorylation along patatin spots seems to be genuine and cannot be explained only by changes in protein abundance.

The control of tuber sprouting is a major target in potato breeding because premature tuber sprouting during their lengthy storage leads to important quality and economic loss [41–43]. However, the molecular mechanisms controlling dormancy release and tuber sprouting are not yet sufficiently known [42–44]. The identification and mapping of phosphorylated isoforms Identification and Mapping of Phosphorylated Isoforms of the Major Storage Protein of Potato... http://dx.doi.org/10.5772/intechopen.70400 77

**Figure 4.** Evaluation of the differential *PR* along 2-DE patatin spots (mature tuber). (a) UPGMA dendrogram from the matrix of mean differences in *PR* between pairs of patatin spots. Spot numbers refer to numbers in **Figure 1**. (b) Mean *PR* values for each of the three main spot groups clustered by UPGMA. Bootstrapping (2000 replicates) was used to determine 99% CIs for mean *PR*-values at each group. *PR*-values over spots were calculated using the formula *PR* = [(*T* − *D*)/*T*] × 100, where *T* and *D* represent the gel-spot volume in reference and dephosphorylated patatin profiles, respectively. Spot volumes over quadruplicate 2-DE gels were determined using the PDQuest software.

hypothesis, isoforms located on acidic gel positions tended to be more highly phosphorylated than those of basic positions. It is also noteworthy that phosphorylation levels were estimated using the measure *PR*, which takes into account the amount of protein at each spot. Therefore, differential phosphorylation along patatin spots seems to be genuine and cannot be explained

**Table 2.** Mean (±SE) values of *PR* for patatin spots estimated from four replicates from dormant tubers of cv. Kennebec.

Volume of spots for untreated and dephosphorylated protein samples with HF-P were assessed by PDQuest software.

are the bootstrap mean and the sample mean estimates, respectively.

∧ *<sup>B</sup>* ≤ *θ* ∧

) ≠ 0.50, which was calculated from 2000

**P (** *θ* ^  *<sup>B</sup>***≤***θ* ^  **)b**

 4.84 39.75 ± 2.53 0.53 35.6, 44.1 34.6, 44.3 4.88 41.21 ± 6.07 0.57 31.9, 52.9 30.1, 54.5 4.90 43.05 ± 2.88 0.55 37.5, 47.9 37.4, 49.3 4.93 28.11 ± 4.35 0.76 23.8, 32.5 23.8, 32.5 4.96 42.83 ± 2.02 0.56 40.1, 46.9 39.4, 48.3 4.96 52.34 ± 4.10 0.57 46.4, 60.4 44.5, 61.8 5.02 39.01 ± 4.11 0.51 32.4, 45.6 30.4, 45.7 5.02 27.19 ± 5.61 0.52 16.9, 35.0 15.3, 35.0 5.05 32.48 ± 3.30 0.51 26.7, 37.3 22.9, 38.0 5.12 30.48 ± 4.72 0.57 23.3, 40.2 22.6, 42.4 5.12 25.13 ± 4.31 0.57 18.8, 33.1 16.6, 34.1 5.13 51.39 ± 5.52 0.55 41.2, 60.6 40.0, 62.1 5.14 4.60 ± 2.04 0.75 2.6, 6.6 2.6, 6.6 5.16 44.34 ± 9.37 0.52 25.7, 57.3 25.0, 59.0 5.20 41.87 ± 3.80 0.52 35.7, 49.3 34.7, 51.2 5.20 26.99 ± 8.37 0.58 18.1, 37.0 10.4, 37.0 5.23 35.96 ± 2.42 0.53 32.9, 40.9 32.2, 42.8 5.25 34.16 ± 4.09 0.53 26.2, 39.6 25.6, 40.0 5.29 N/A<sup>d</sup> N/A N/A N/A 5.27 11.95 ± 4.63 0.55 6.8, 20.9 5.6, 20.9

**95% bootstrap CI (CL, CU)c**

**99% bootstrap CI (CL, CU)c**

The control of tuber sprouting is a major target in potato breeding because premature tuber sprouting during their lengthy storage leads to important quality and economic loss [41–43]. However, the molecular mechanisms controlling dormancy release and tuber sprouting are not yet sufficiently known [42–44]. The identification and mapping of phosphorylated isoforms

only by changes in protein abundance.

Gel position of assigned spots is shown in **Figure 1**.

∧ *<sup>B</sup>* and *θ* ∧

The bootstrap distribution is median biased if the probability (P) of ( *θ*

<sup>d</sup>N/A = not available, weakly stained spot with a volume below the limit of detection.

CI—Confidence interval; CL—lower bound; CU—upper bound.

a

b

c

bootstrap replicates; *θ*

**Spot no.a p***I* **Mean (± SE)** *PR*

76 Advances in Seed Biology

of the patatin opens up new exploratory ways to unravel the molecular mechanisms underlying mobilization of VSPs. The finding of differentially phosphorylated isoforms is particularly relevant because increase (or decrease) in phosphorylation status without a parallel change in the amount of protein has been considered to be a useful indicator for a specific functional change [39, 40, 45]. In this regard, systematic follow-up studies on VSPs will be needed to assess whether their degradation takes place through a phosphorylation-dependent regulatory mechanism, as it occurs in common bean during dry-to-germinating seed transition [10]. The establishment of a 2-DE-based reference map of patatin can be a very efficient tool to address this challenge in potato by monitoring changes in the phosphorylation status along the tuber life cycle.
